Anti-Interferon Alpha Monoclonal Antibodies and Methods For Use

ABSTRACT

The present invention includes compositions and methods that include antibodies that selectively neutralize a bioactivity of at least two interferon alpha (“IFNα”) protein subtypes for the protein subtypes A, 2, B2, C, F, G, H2, I, J1, K, 4a, 4b and WA, but does not neutralize at least one bioactivity of IFNα protein subtype D. Examples of bioactivity for measurement include activation of the MxA promoter or antiviral activity and variants, derivatives and fragments thereof. The invention also includes host cells, hybridomas and plasmacytomas that produce antibodies. Because of their unique selectivity and affinity, the antibodies of the present invention are useful to detect IFNα subtypes in sample or tissue and/or for therapeutic applications that include, but are not limited to the treatment and/or amelioration of an IFNα related disorder such as SLE, lupus, type I diabetes, psoriasis, AIDS and Graft versus Host Disease.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of International Application No.PCT/US2007/075616, filed Aug. 9, 2007, which claims the benefit of U.S.Provisional Application No. 60/836,599, filed Aug. 9, 2006. Thisapplication is also a continuation-in-part of U.S. application Ser. No.11/883,961, filed Aug. 8, 2007, which is a National Stage application ofInternational Application No. PCT/US2006/004643, filed Feb. 9, 2006,which claims the benefit of U.S. Provisional Application No. 60/652,233,filed Feb. 10, 2005. The entire contents of all of the foregoingapplications are hereby incorporated by reference herein.

FIELD OF THE INVENTION

The present invention relates to methods and compositions useful todiagnose and treat conditions correlated with an abnormal level ofinterferon-α (IFNα) expression in a subject.

BACKGROUND OF THE INVENTION

Human interferons (IFNs) are functionally-related cytokines thatmodulate both innate and adaptive immune responses. They are categorizedin two groups, largely on the basis of sequence homology, as Type I andII. Type I IFNs include six types (IFN-α, IFN-β, IFN-ω, IFN-κ, IFN-ε,and IFN-λ). IFN-α, β, ω, and κ act through an identical IFN receptor,IFNAR. IFN-λ associates with a distinct receptor, IFNLR. The receptorfor IFN-ε is currently unknown. Type II IFN consists of a single type(IFN-γ) and associates with the receptor IFNGR. While Type I IFNs arestrongly induced during viral infections, Type II IFN is inducedprimarily in response to immune and inflammatory stimuli, and thus IFN-γis frequently referred to as “immune IFN”. The most studied of thenumerous Type I IFNs include IFN-α, IFN-β, and IFN-ω. Of these, IFN-α isthe most complex, includes at least fifteen distinct protein subtypesexhibiting upwards of 75% sequence homology. Diaz (1995) Semin. Virol.6:143-149; Weissmann et al. (1986) Prog Nucl Acid Res Mol Biol 33:251; JInterferon Res (1993) 13:443-444; Roberts et al. (1998) J InterferonCytokine Res 18:805-816. In addition to having structural similarity,the IFN-α genes and their products show functional similarities. Forexample, they are induced by dsRNA or virus, and can interact with thesame receptor, the IFNα/β receptor IFNAR. Mogensen, et al. (1999) J.Interferons and other Regulatory Cytokines, John Wiley & Sons. IFNα alsoinhibits apoptosis, promotes the survival and differentiation ofantigen-activated T helper cells and promotes the maturation offunctionally efficient monocyte-derived dendritic cells.

Many types of cells produce IFNα when exposed to viruses and dsRNA.Specialized leukocytes (called interferon-α producing cells (“IPCs”)produce IFNα in response to a wider variety of stimuli, e.g., viruses,bacteria and protozoa. Several in vitro studies indicate that thevarious IFN-α subtypes are produced to different extents by distinctIFN-α-secreting cell lines or in a virus type-specific manner followinginfection of human peripheral blood mononuclear cells (PBMCs), and thatthese patterns are often associated with subtype-dependent differencesin anti-proliferative, anti-viral, and anti-tumor activities. However,the physiological significance of the individual subtypes and theirsynergistic or antagonist activities with one another in vivo remainsundefined.

IFN-α has been implicated as a mediator of the pathology seen in severalautoimmune diseases. Moreover, it can cause autoimmune diseasedevelopment in patients treated with IFN-α for cancer and viralinfections. Increased expression of IFN-α has been observed in thedisease-localized tissues of patients with insulin-dependent diabetesmellitus (IDDM or type I diabetes), psoriasis, Crohn's disease, andceliac disease. Over expression of IFN-α has been observed in patientswith systemic lupus erythematosus (SLE), IDDM and AIDS. In the case ofSLE, which is characterized by an abundance of both autoreactive B and Tcells, IFN-α expression is observed in not only tissue lesions butcirculating within the blood of afflicted individuals. Furthermore, theIFN-α serum levels tend to correlate with the clinical disease activityindex. This is believed to stem from cyclical induction of normallyquiescent monocytes into potent antigen-presenting dendritic cells (DCs)as triggered by upregulation of IFN-α production by plasmacytoid DCs(pDCs). Indeed, the present inventors have previously demonstrated viaoligonucleotide microarray analysis that SLE can be distinguished by“signatures” of unregulated genes involved in granulopoiesis and IFNinduction; these signatures revert to normal upon high-dose infusion ofglucocorticoids (U.S. patent application Ser. No. 11/228,586, thecontent of which is incorporated by reference hereto).

Systemic lupus erythematosus (SLE) is a systemic autoimmune rheumaticdisease that is particularly aggressive in children and characterized byflares of high morbidity. Autoimmune diseases such as SLE often act inself-perpetuating cycles of relapse and remission. These cycles areoften defined by phases of treatment with generally therapeutic regimensadministered to quench the SLE disease cycle. FDA-approved treatmentoptions for SLE include corticosteroids, nonsteroidal immunesuppressants, antimalarials, and nonsteroidal anti-inflammatory drugs.These drugs abrogate the integrity of all immune effector responsesrather than acting upon those specific to the pathogenesis of SLE. SLErepresents an unmet medical need since these treatments are onlypartially effective with moderate to severe side-effects including bonethinning, weight gain, acne, anemia, sterility, rashes diarrhea, hairloss, and nausea. Furthermore, no new therapeutics for SLE have beenapproved in 40 years.

SLE has recently been closely linked to unabated IFNα production. Shi etal. (1987) Br. J. Dermatol. 117(2):155-159. IFNα is present at elevatedlevels in SLE serum (Crow et al. (2004) Curr. Opin. Rheumatol.16(5):541-547) and plasmacytoid DCs (pDCs), the primary source of IFNα,accumulate in SLE skin. Farkas et al. (2001) Am. J. Pathol.159(1):237-243. Moreover, it has been observed that some patientstreated with IFNα have developed lupus (Okanoue et al. (1996) J.Hepatol. 25(3):283-291; Tothova et al. (2002) Neoplasma 49(2):91-94; andRaanani et al. (2002) Acta Haematol. 107(3):133-144) and that lupuspatients that present with IFNα antibodies have been shown to display amilder form of the disease. Von Wussow et al. (1988) Rheumatol. Int.8(5):225-230. IFNα may act via the differentiation of monocytes intofunctional dendritic cells (DCs) which in turn mediates theetiopathogenesis of SLE. Pascual V. et al. (2003) Curr. Opin. Rheumatol.15(5):548-556. A proposed approach for the treatment of SLE isneutralization of IFNα (see Banchereau et al., PCT/US02/00343, thecontents of which is incorporated by reference).

Although monoclonal antibodies (MAbs) that can block human IFN-αbioactivity have been produced, none have been reported to date that canneutralize all fifteen known subtypes, and few can neutralizenaturally-derived, IFN-α-containing leukocyte IFN. PBL BiomedicalLaboratories offers ten mouse monoclonal antibodies that bind tomultiple human IFNα gene subtypes (see the world wide web atinterferonsource.com/relativespecificity.html). However, each of the PBLantibodies bind the IFNα protein subtype encoded by human IFNα genesubtype 1 (IFNα protein subtype D) and from up to one to twelve otherIFNα subtypes. U.S. Patent Publ. No. 2003/0166228A1 discloses amonoclonal antibody (designated 9F3) that was derived from immunizationof mice with leukocyte IFNα (which includes all of the IFNα proteinsubtypes). The 9F3 MAb binds and neutralizes the anti-viral activity ofthe proteins encoded by seven human IFNα gene subtypes 1, 2, 4, 5, 8, 10and 21 (which encode IFNα protein subtypes D, A, 4, G, B2, C and F,respectively), without neutralizing the antiviral activity of humanIFNβ. This publication does not disclose whether the 9F3 antibody bindsand inactivates the other eight IFNα gene subtypes, nor whether it bindsto one or both of IFNα protein subtypes 4a and 4b. The PCT publicationsuggests using the monoclonal antibodies to treat disorders associatedwith increased expression of IFNα's, in particular, autoimmune disorderssuch as insulin-dependent diabetes mellitus and SLE. However, it is notknown whether the 9F3 antibody is able to sufficiently neutralize thebiological activity of the IFNα protein subtypes found in SLE serum.

Because IFNα is a multi-functional mediator of the immune response andhas beneficial antiviral activity, complete inhibition or significantdown-regulation of all IFNα subtypes is not an optimal therapeuticapproach. Thus, a need exists for agents that will selectivelyneutralize the IFNα subtypes associated with pathological conditions.This invention satisfies this need and provides related advantages aswell.

SUMMARY OF THE INVENTION

The invention provides monoclonal antibodies and derivatives thereofthat neutralize specific IFNα subtypes. The antibodies of the inventionare useful in the amelioration and treatment of conditions associatedwith increased expression of IFNα, such as SLE, psoriasis, type Idiabetes, AIDS, Graft versus Host Disease and other autoimmune diseases.In one aspect, the invention includes an antibody that selectivelyneutralizes a bioactivity of at least two interferon alpha (“IFNα”)protein subtypes such as subtypes A, 2, B2, C, F, G, H2, I, J1, K, 4a,4b and WA, but does not significantly neutralize at least onebioactivity of IFNα protein subtype D; wherein the bioactivity isactivation of the MxA promoter or antiviral activity. The antibodypreferably binds essentially the same, or the same, or competes with anantibody that binds essentially the same, or the same, IFNα epitope asthe anti-IFNα antibody produced by the hybridoma having ATCC AccessionNo. PTA-7778. Preferably, the antibody is a monoclonal antibody. In oneembodiment, the antibody does not significantly neutralize IFNα proteinsubtype D nor 1. In one embodiment, the monoclonal antibody is ACO-2.Preferably, the antibodies of the invention inactivate the ability ofserum isolated from systemic lupus erythematosus (SLE) patients tostimulate the differentiation of monocytes into dendritic cells.

In another aspect, a monoclonal anti-interferon alpha (“IFNα”) antibodyproduced by the hybridoma having ATCC Accession No. PTA-7778 isprovided.

The antibodies, derivatives or fragments thereof of the invention can bemouse, rat, human, or from other mammals, or fragments or humanized orchimeric forms thereof. The antibodies of the invention include mousemonoclonal antibodies, e.g., ACO-2, as well as humanized forms, chimericforms, or fragments thereof. The invention further provides antibodiesthat bind to essentially the same IFNα epitope as murine monoclonalantibody ACO-2. The invention further provides host cells, hybridomas,compositions, pharmaceutical compostions and kits comprising theantibodies of the invention.

The antibodies, derivatives or fragments thereof of the invention haveuse in the treatment of diseases or conditions associated withoverexpression of IFNα, including but not limited to SLE, psoriasis,AIDS, type I diabetes and autoimmune thyroiditis, and for the productionof medicaments to treat such diseases and conditions. The antibodies ofthe invention can also be used to distinguish or to purify various IFNαsubtypes.

The invention also includes host cells and hybridoma cell lines thatproduce antibodies having the above-noted specificities, e.g., specificbinding to one or more interferon alpha (“IFNα”) protein subtypes (A, 2,B2, C, F, G, H2, I, J1, K, 4a, 4b and WA), but does not neutralize thebioactivity of, e.g., IFNα protein subtype D. The antibodies and/orhybridoma cell lines can be combined with a carrier, such as apharmaceutically acceptable carrier, for use in diagnostic andtherapeutic methods. The antibodies are useful to detect specific IFNαsubtypes and to diagnose, prognose, treat and/or ameliorate symptoms ofIFNα related disorders. Examples of such conditions include, but are notlimited to SLE, psoriasis, type I diabetes, Graft versus Host (GVH)Disease, AIDS, autoimmune thyroiditis and other autoimmune disorders.The antibodies of the invention are also useful to neutralize and/orisolate these IFNα subtypes in vitro or in vivo.

BRIEF DESCRIPTION OF THE FIGURES

For a more complete understanding of the features and advantages of thepresent invention, reference is now made to the detailed description ofthe invention along with the accompanying figures and in which:

FIG. 1 shows a schematic diagram of the Reporter Gene (RG) assay and theCytopathic Effect Inhibition Assay (CPE). The filled circles in the CPEassay diagram represent live, intact cells. The unfilled circlesrepresent dead cells, killed by viral infection. The filled circles andcells as well as the “+” in the RG assay diagram represent luciferaseexpression, while the unfilled circles and cells represent the lack ofluciferase expression.

FIG. 2 shows a flow chart of the IFNα MAb development scheme.

FIG. 3 shows neutralization of complex IFN sources by ACO-1, 2, 3, 4,and 5. (a) Neutralization of 600 pg of leukocyte IFN (Sigma) by theindicated amounts of each MAb was evaluated via the RG bioassay.Blockade percentages were calculated based upon the LCPS values obtainedin the presence/absence of leukocyte IFN and the absence of any MAb.Values represent the mean of triplicates. (b) Neutralization of PBMC-flu(640-fold dilution) by each MAb. Blockade percentages were calculated aspreviously described. Values represent the mean of triplicates.

FIG. 4 shows neutralization of fifteen recombinant IFN-α subtypes byACO-1. (a) Neutralization of the indicated IFN-α subtypes by increasingconcentrations of ACO-1 was evaluated via the RG bioassay. The numericalvalue assigned to each curve represents the midpoint (EC₅₀) calculatedfrom the LCPS value (luminescence counts per second) obtained in theabsence of ACO-1 (indicated by open circles on the Y-axis) and thehighest concentration of MAb tested (2000 ng/ml). N.D. signifies that noEC₅₀ value could be assigned. Data points were derived from triplicates.(b) Lack of neutralization of IFN-β by increasing concentrations ofACO-1, 2, 3, 4, 5, and 8. Data points were derived from triplicates.

FIG. 5 shows the results of a multiplex analysis of monoclonalantibodies ACO-1, ACO-2, ACO-3, ACO-4, ACO-5, and ACO-6.

FIG. 6 shows the results of solid-phase binding assay of IFNα subtypesby monoclonal antibodies ACO-1, ACO-2, ACO-3, ACO-4, ACO-5, and ACO-6.

FIG. 7 shows the neutralization of SLE patient serum samples SLE-43 (a),SLE-133 (b), SLE-140 (c), and SLE-BD (d) bioactivity evaluated by theCPE assay. Amounts of MAb tested are indicated. Controls include serumalone, media only (−), and a pan-neutralizing polyclonal antibody (pAb,rabbit and anti-human IFNα, PBL). Values represent the mean oftriplicates.

FIG. 8A-C shows the cross-reactivity of ACO-1 (A), ACO-2 (B), and ACO-3(C) with 156 pg/well Macaque IFN.

FIG. 9 shows the cDNA and amino acid sequence of the heavy chain fromACO-1. The DNA sequence encoding the V_(H)1, V_(H)2 and V_(H)3 CDRs areshown in italics, while the corresponding amino acid sequences areunderlined.

FIG. 10 shows the cDNA and amino acid sequence of the light chain fromACO-1. The DNA sequence encoding the V_(L)1, V_(L)2 and V_(L)3 CDRs areshown in italics, while the corresponding amino acid sequences areunderlined.

DETAILED DESCRIPTION OF THE INVENTION

While the making and using of various embodiments of the presentinvention are discussed in detail below, it should be appreciated thatthe present invention provides many applicable inventive concepts thatcan be embodied in a wide variety of specific contexts. The specificembodiments discussed herein are merely illustrative of specific ways tomake and use the invention and do not delimit the scope of theinvention.

To facilitate the understanding of this invention, a number of terms aredefined below. Terms defined herein have meanings as commonly understoodby a person of ordinary skill in the areas relevant to the presentinvention. Terms such as “a”, “an” and “the” are not intended to referto only a singular entity, but include the general class of which aspecific example may be used for illustration. The terminology herein isused to describe specific embodiments of the invention, but their usagedoes not delimit the invention, except as outlined in the claims. Forexample, the term “a cell” includes a plurality of cells, includingmixtures thereof. All numerical designations, e.g., pH, temperature,time, concentration, and molecular weight, including ranges, areapproximations which are varied (+) or (−) by increments of 0.1. It isto be understood, although not always explicitly stated that allnumerical designations are preceded by the term “about”. It also is tobe understood, although not always explicitly stated, that the reagentsdescribed herein are merely exemplary and that equivalents of such areknown in the art.

The term “antibody”, as used herein, refers to all classes andsubclasses of intact immunoglobulins. The term “antibody” also coversmonoclonal antibodies, antibody fragments and antibody fragment clones.“Antibody fragments” include a portion of an intact antibody thatcontains the antigen binding or variable region of the intact antibody.Examples of antibody fragments include Fab, Fab′, F(ab′)2, and Fvfragments; single-chain antibody molecules, multispecific antibodiesformed from antibody fragments; a Fd fragment includes the VH and CH,domains; a Fv fragment includes the VL and VH domains of a single arm ofan antibody, a dAb fragment (Ward et al. (1989) Nature 341:544-546),which includes a VH domain; and an isolated complementarity determiningregion (CDR). “Single-chain Fv” or “scFv” antibody fragments include theVH and VL domains of antibody, wherein these domains are present in asingle polypeptide chain. Generally, the scFv polypeptide furtherincludes a polypeptide linker between the VH and VL domains, whichenables the scFv to form the desired structure for antigen binding. Fora review of scFv see Pluckthun (1994) in The Pharmacology of MonoclonalAntibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, NewYork, pp. 269-315, Dall'Acqua and Carter (1998) Curr. Opin. Struct.Biol. 8: 443-450, Hudson (1999) Curr. Opin. Immunol. 11: 548-557, Birdet al. (1988) Science 242:423-426 and Huston et al. (1988) Proc. Natl.Acad. Sci. USA 85:5879-5883. Any of the above-noted antibody fragmentsmay be obtained using conventional techniques known to those of skill inthe art, and the fragments are screened for binding specificity andneutralization activity in the same manner as are intact antibodies. Theantibodies can be isolated from any suitable biological source, e.g.,murine, rabbit, rat, human, sheep, canine, etc. “Naturally occurring” or“native” antibodies are heterotetrameric glycoproteins, typically havinga molecular weight of approximately 150-200 kD. The heterotetramerincludes two identical light (L) chains and two identical heavy (H)chains. Each light chain is covalently bonded to a heavy chain by adisulfide bond.

The term “antibody derivative”, as used herein, refers to encompassmolecules that bind an epitope and which are modifications orderivatives of a native monoclonal antibody of this invention.Derivatives include, but are not limited to, for example, bispecific,multispecific, heterospecific, trispecific, tetraspecific, multispecificantibodies, chimeric, recombinant and humanized.

The term “antibody variant, as used herein, refers to antibodiesproduced in a species other than a mouse or an isotype of an antibodyselected from the antibodies designated ACO-1 through ACO-6 and ACO-8.The term “antibody variant” also includes antibodies containingpost-translational modifications to the linear polypeptide sequence ofthe antibody or fragment. It further encompasses fully human antibodies.

The term “monoclonal antibody”, as used herein, refers to an antibody(including antibody fragments) obtained from a population ofsubstantially homogeneous antibodies, i.e., the individual antibodies inthe population are identical except for possible naturally occurringmutations that may be present in minor amounts, which are also part ofthe present invention so long as they exhibit the desired biologicalactivity. Monoclonal antibodies are highly specific and directed againsta single epitope. Monoclonal antibodies may be synthesized by ahybridoma culture, in a bio-reactor, as an ascites or made byrecombinant methods, such as in vitro translation, in bacteria, yeast,plants, insect and/or animal cells. Accordingly, the modifier“monoclonal” indicates the character of the antibody as being obtainedfrom a substantially homogeneous population of antibodies, and is not tobe construed as requiring production of the antibody by any particularmethod. For example, the monoclonal antibodies to be used in accordancewith the present invention may be made by the hybridoma method firstdescribed by Kohler et al. (1975) Nature, 256:495, or may be made byrecombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567).“Monoclonal antibodies” includes, but is not limited to human monoclonalantibodies, humanized monoclonal antibodies, recombinant humanantibodies, clones of antigen-recognition and binding-site containingantibody fragments (Fv clones) isolated from phage antibody librariesand derivatives thereof. (See Clackson, et al. (1991) Nature,352:624-628; and Marks, et al. (1991) J Mol Biol 222:581-597).Monoclonal antibodies also include “chimeric” antibodies(immunoglobulins) in which, e.g., a portion of the heavy and/or lightchain is identical with or homologous to corresponding sequences inantibodies derived from a particular species or belonging to aparticular antibody class or subclass, while the remainder of thechain(s), or portions thereof, is identical with or homologous tocorresponding sequences in antibodies derived from another species orbelonging to another antibody class or subclass, as well as fragments ofsuch antibodies, so long as they exhibit the desired biological activity(U.S. Pat. No. 4,816,567; Morrison, et al., (1984) Proc Natl Acad SciUSA, 81:6851-6855).

The term “human monoclonal antibody”, as used herein, refers toantibodies displaying a single binding specificity that have variableand constant regions derived from human germline immunoglobulinsequences.

The term “human antibody”, as used herein, refers to antibodies havingvariable and constant regions derived from human germline immunoglobulinsequences. The human antibodies of the invention may include amino acidresidues not encoded by human germline immunoglobulin sequences (e.g.,mutations introduced by random or site-specific mutagenesis in vitro orby somatic mutation in vivo). However, the term “human antibody” as usedherein, is not intended to include antibodies in which CDR sequencesderived from the germline of another mammalian species, such as a mouse,have been grafted onto human framework sequences. Thus, as used herein,the term “human antibody” refers to an antibody in which substantiallyevery part of the protein (e.g., CDR, framework, CL, CH domains (e.g.,CH₁, CH₂, CH₃), hinge, (VL, VH)) is substantially non-immunogenic inhumans, with only minor sequence changes or variations. Similarly,antibodies designated primate (monkey, baboon, chimpanzee, etc.), rodent(mouse, rat, rabbit, guinea pig, hamster, and the like) and othermammals designate such species, sub-genus, genus, sub-family, familyspecific antibodies. As described hereinabove, chimeric antibodies mayalso include any combination of the above. Such changes or variationsoptionally and preferably retain or reduce the immunogenicity in humansor other species relative to non-modified antibodies. Thus, a humanantibody is distinct from a chimeric or humanized antibody. A humanantibody may be produced by a non-human animal or prokaryotic oreukaryotic cell that is capable of expressing functionally rearrangedhuman immunoglobulin (e.g., heavy chain and/or light chain) genes.Further, when a human antibody is a single chain antibody, it may alsoinclude a linker peptide that is not found in native human antibodies.For example, an Fv fragment may also include a linker peptide, such astwo to about eight glycine or other amino acid residues, which connectsthe variable region of the heavy chain and the variable region of thelight chain. Such linker peptides are considered to be of human origin.

A human antibody is “derived from” a particular germline sequence if theantibody is obtained from a system using human immunoglobulin sequences,e.g., by immunizing a transgenic mouse carrying human immunoglobulingenes or by screening a human immunoglobulin gene library. A humanantibody that is “derived from” a human germline immunoglobulin sequencecan be identified as such by comparing the amino acid sequence of thehuman antibody to the amino acid sequence of human germlineimmunoglobulins. A selected human antibody typically is at least 90%identical in amino acids sequence to an amino acid sequence encoded by ahuman germline immunoglobulin gene and contains amino acid residues thatidentify the human antibody as being human when compared to the germlineimmunoglobulin amino acid sequences of other species (e.g., mouse or ratgermline sequences). In certain cases, a human antibody may be at least95%, or even at least 96%, 97%, 98%, or 99% identical in amino acidsequence to the amino acid sequence encoded by the germlineimmunoglobulin gene. Typically, a human antibody derived from aparticular human germline sequence will display no more than 10 aminoacid differences from the amino acid sequence encoded by the humangermline immunoglobulin gene. In certain cases, the human antibody maydisplay no more than 5, or even no more than 4, 3, 2, or 1 amino aciddifference from the amino acid sequence encoded by the germlineimmunoglobulin gene.

The term “humanized”, as used herein, refers to the use of portions of anon-human (e.g., mouse or rat) antibodies that are used on a humanimmunoglobulin backbone to make chimeric immunoglobulins, immunoglobulinchains or fragments thereof (such as Fv, Fab, Fab′, F(ab′)2 or otherantigen-binding subsequences of antibodies) that have sequences derivedfrom non-human immunoglobulin. Generally, humanized antibodies are humanimmunoglobulins (recipient antibody) in which residues from part or allof one or more complementarity-determining regions (CDRs) of therecipient antibody are replaced by residues from one or more CDRs of anon-human species (donor antibody) such as mouse, rat or rabbit havingthe desired specificity, affinity, and capacity. For example, Fvframework region (FR) residues of the human immunoglobulin are replacedby corresponding non-human residues, or vice-versa, that is, humanimmunoglobulin portions may be grafted onto the non-human immunoglobulinregions that determine antigen specificity. Furthermore, humanizedantibodies may include residues that are found neither in the recipientantibody, nor in the imported CDR or framework sequences. Themodifications that are not part of the donor or recipient antibody arecommonly and easily made to further refine and optimize antibodyperformance. In general, the humanized antibody will includesubstantially all of at least one, and typically both, variable domains(light and heavy), in which all or substantially all of the CDR regionscorrespond to those of a non-human immunoglobulin and all orsubstantially all of the FR regions are those of a human immunoglobulinsequence. The humanized antibody may also include at least a portion ofan immunoglobulin constant region (Fc), typically that of a humanimmunoglobulin.

The term “recombinant human antibody”, as used herein, includes allhuman antibodies that are prepared, expressed, created or isolated byrecombinant method, such as antibodies isolated from an animal (e.g., amouse) that is transgenic or transchromosomal for human immunoglobulingenes or a hybridoma prepared therefrom, antibodies isolated from a hostcell transformed to express the antibody, e.g., from a cell transfectedto express the antibody (commonly a plasmacytoma), antibodies isolatedfrom a recombinant, combinatorial human antibody library, and antibodiesprepared, expressed, created or isolated by any other method that mayinvolve splicing of human immunoglobulin gene sequences to other DNAsequences. Such recombinant human antibodies have variable and constantregions derived from human germline immunoglobulin sequences. In certainembodiments, however, such recombinant human antibodies can be subjectedto in vitro mutagenesis (or, when an animal transgenic for human Igsequences is used, in vivo somatic mutagenesis) and thus the amino acidsequences of the VH and VL regions of the recombinant antibodies aresequences that, while derived from and related to human germline VH andVL sequences, may not naturally exist within the human antibody germlinerepertoire in vivo.

The terms “antigen-binding site” or “binding portion”, as used herein,refer to the part of an immunoglobulin molecule that participates inantigen binding. The antigen binding site is formed by amino acidresidues of the N-terminal three variable (“V”) regions of the heavy(“H”) chain and three variable regions of light (“L”) chains. Threehighly divergent stretches within the V regions of the heavy and lightchains are referred to as “hypervariable regions” or “CDRs”, of whichthree are interposed between four conserved flanking stretches known as“framework regions” (FR). Framework regions refer to amino acidsequences that are found naturally between, and adjacent to,hypervariable regions in immunoglobulins. In antibody molecules, thethree hypervariable regions of a light chain (V_(L)1, V_(L)2 and V_(L)3)and the three hypervariable regions of a heavy chain (V_(H)1, V_(H)2 andV_(H)3) are disposed relative to each other in three dimensional spaceto form an antigen-binding surface. The antigen-binding surface iscomplementary to the three-dimensional surface of a bound antigen, andthe three hypervariable regions of each of the heavy and light chainsare referred to as “complementarity-determining regions” or “CDRs.”

The term “bioactivity”, as used herein, refers to the ability of one ormore IFNα subtypes (or IFNβ) to activate the MxA promoter (andinterferon-inducible promoter) or to exert an antiviral effect. The EC₅₀and percent neutralization for an antibody of an IFNα bioactivity canvary depending on the assay conditions and the type of IFNα bioactivitymeasured. For consistency, specific types of bioactivities (i.e.,activation of the M_(x)A promoter and antiviral activity) and assayconditions (i.e., the “RG assay” and the “CPE assay”) are used. The RGassay can be performed using the conditions described herein. Percentneutralization of activation of the M_(x)A promoter is determined asdescribed in the Examples (see Example 3), using RGmax IFN amounts and 2micrograms per mL antibody. The antiviral (CPE) assay can be performedaccording to the methods described in the examples.

The term “bispecific molecule”, as used herein, refers to any agent,e.g., a protein, peptide, or protein or peptide complex, which has twodifferent binding specificities. The term “multispecific molecule” or“heterospecific molecule” is intended to include any agent, e.g. aprotein, peptide, or protein or peptide complex, which has more than twodifferent binding specificities.

An antibody binds “essentially the same epitope” as a reference antibodywhen the two antibodies recognize identical or sterically overlappingepitopes. Antibody binding may be measured or determined by standardantibody-antigen assays, for example, competition assays, saturationassays, or standard immunoassays such as ELISA or RIA. The most widelyused and rapid methods for determining whether two antibodies bind toidentical or sterically overlapping epitopes are competition assays,which can be configured in a number of different formats using eitherlabeled antigen or labeled antibody. Usually, the antigen is immobilizedon a 96-well plate, and the ability of unlabeled antibodies to block thebinding of labeled antibodies is measured using radioactive or enzymelabels. One example of a competition ELISA assay is disclosed in U.S.Pat. No. 5,512,457. Other assays are known in the art for how todetermine whether antibodies bind to the same or substantially oressentially the same epitope as a given antibody or that effectivelycompete with a given antibody for binding to an antigen, for examplethose described in U.S. Pat. Nos. 6,342,219, 6,342,221, 6,524,583,6,416,758 and U.S. Patent Publication No. 2007/0065447, each of whichare incorporated herein by reference.

The term “composition”, as used herein, refers to a combination ofactive agent and another carrier, e.g., compound or composition, inert(for example, a detectable agent or label) or active, such as anadjuvant, diluent, binder, stabilizer, buffers, salts, lipophilicsolvents, preservative, adjuvant or the like. Carriers also includepharmaceutical excipients and additives proteins, peptides, amino acids,lipids, and carbohydrates (e.g., sugars, including monosaccharides, di-,tri-, tetra-, and oligosaccharides; derivatized sugars such as alditols,aldonic acids, esterified sugars and the like; and polysaccharides orsugar polymers), which can be present singly or in combination,including alone or in combination 1-99.99% by weight or volume.Exemplary protein excipients include serum albumin such as human serumalbumin (HSA), recombinant human albumin (rHA), gelatin, casein, and thelike. Representative amino acid/antibody components, which can alsofunction in a buffering capacity, include, e.g., alanine, glycine,arginine, betaine, histidine, glutamic acid, aspartic acid, cysteine,lysine, leucine, isoleucine, valine, methionine, phenylalanine,aspartame, and the like. Carbohydrate excipients are also intendedwithin the scope of this invention, examples of which include but arenot limited to monosaccharides such as fructose, maltose, galactose,glucose, D-mannose, sorbose, and the like; disaccharides, such aslactose, sucrose, trehalose, cellobiose, and the like; polysaccharides,such as raffinose, melezitose, maltodextrins, dextrans, starches, andthe like; and alditols, such as mannitol, xylitol, maltitol, lactitol,xylitol sorbitol (glucitol) and myoinositol. The term carrier furtherincludes a buffer or a pH adjusting agent; typically, the buffer is asalt prepared from an organic acid or base. Representative buffersinclude organic acid salts such as salts of citric acid, ascorbic acid,gluconic acid, carbonic acid, tartaric acid, succinic acid, acetic acid,or phthalic acid; Tris, tromethamine hydrochloride, or phosphatebuffers. Additional carriers include polymeric excipients/additives suchas polyvinylpyrrolidones, ficolls (a polymeric sugar), dextrates (e.g.,cyclodextrins, such as 2-hydroxypropyl-.quadrature.-cyclodextrin),polyethylene glycols, flavoring agents, antimicrobial agents,sweeteners, antioxidants, antistatic agents, surfactants (e.g.,polysorbates such as “TWEEN 20” and “TWEEN 80”), lipids (e.g.,phospholipids, fatty acids), steroids (e.g., cholesterol), and chelatingagents (e.g., EDTA).

The term “control”, as used herein, refers to is an alternative subjector sample used in a study for comparison purpose. A control can be“positive” or “negative”.

The term “effective amount”, as used herein, refers to is an amountsufficient to effect beneficial or desired results. An effective amountcan be administered in one or more administrations, applications ordosages.

The terms “epitope” or “antigenic determinant”, as used herein, refer toa site on an antigen, or an antigen fragment, recognized by an antibodyor an antigen receptor. A T cell epitope is a short peptide derived froma protein antigen that is presented by the appropriate MajorHistocompatibility Compatibility (MHC) protein. B-cell epitopes aregenerally antigenic determinants recognized by B cells and are commonlyportions of a three-dimensional surface that are recognized by anantibody, which may include sequential or conformational determinants,as will be known to the skilled artisan.

Antigenic determinant regions and predicted epitopes for the IFNαantibodies may be identified by any method for determining antigenicdeterminant regions, such as standard mapping and characterizationtechniques, further refinement of which can be accomplished byapplication of any suitable technique (references to “epitope mapping”techniques, “epitope identification” techniques, and the like, herein,should be understood as describing techniques applicable to theidentification and/or refinement of epitopes). As one example of suchmapping/characterization methods, an epitope for ACO-2 antibody may bedetermined by epitope “footprinting” using chemical modification of theexposed amines/carboxyls in the IFNα protein. One specific example ofsuch a footprinting technique is the use of HXMS (hydrogen-deuteriumexchange detected by mass spectrometry). Briefly, in HXMS, ahydrogen/deuterium exchange of receptor and ligand protein amide protonsis instituted, followed by peptide-antigen binding, and back exchange ofreceptor and ligand protein amide protons. The backbone amide groupsparticipating in protein binding are protected from back exchange andtherefore remain deuterated. Relevant regions can be identified at thispoint by peptic proteolysis, fast microbore high-performance liquidchromatography separation, and/or electrospray ionization massspectrometry. See, e.g., Ehring H, Analytical Biochemistry, Vol. 267 (2)pp. 252-259 (1999) and/or Engen, J. R. and Smith, D. L. (2001) Anal.Chem. 73, 256A-265A. Other methods for epitope mapping include, but arenot limited to, nuclear magnetic resonance (NMR) epitope mapping, massspectrometry methods, protease digestion techniques, and various phagedisplay techniques. Examples of such techniques are described forexample in U.S. Patent Publication No. 2007/0065447, the entirety ofwhich is incorporated herein by reference. Numerous other methods arealso known in the art for epitope mapping, such as those described forexample in O. M. R. Westwood and F. C. Hay, EPITOPE MAPPING: A PRACTICALAPPROACH (Oxford Univ. Press, 2000).

IgG antibodies can be cleaved into three fragments by papain digestion.Two of these fragments are typically identical antigen-bindingfragments, called “Fab” fragments, each with a single antigen-bindingsite, and a residual “Fc” fragment. The Fab fragment contains the lightchain and the amino-terminal half of the heavy chain held together by aninterchain disulfide bond. The Fc fragment consists of thecarboxyterminal halves of the two heavy chains disulfide-bonded to eachother by the residual hinge region. Pepsin digestion of an IgG antibodycleaves in the same general region of the antibody as papain, but on thecarboxy-terminal side of the disulfide bond, to produce the F(ab′)₂fragment, which has two antigen-combining sites and is still capable ofcross-linking antigen. Fab′ fragments differ from Fab fragments by theaddition of a few residues at the carboxy terminus of the heavy chainCH1 domain including one or more cysteines from the antibody hingeregion. Fab′-SH is the designation for Fab′ in which the cysteineresidue(s) of the constant domains bear a free thiol group.

The term “Fv”, as used herein, refers to the minimum antibody fragmentthat includes a complete antigen-recognition and binding site. In atwo-chain Fv species, this region includes a dimer of one heavy- and onelight-chain variable domain in non-covalent association. In asingle-chain Fv species, one heavy- and one light-chain variable domainmay be linked covalently by a flexible peptide linker such that thelight and heavy chains can associate in a “dimeric” structure analogousto that in a two-chain Fv species.

The term “heteroantibodies”, as used herein, refers to two or moreantibodies, antibody binding fragments (e.g., Fab), derivatives thereof,or antigen binding regions linked together that have at least two havedifferent antigen specificities.

The term “Interferon Alpha” (“IFNα”), as used herein, refers to a familyof proteins that include some of the main effectors of innate immunity.There are at least 15 known isotypes of human IFNα. The names of theIFNα protein subtypes and corresponding encoding genes are listed below.

IFNα protein subtype Corresponding IFNα gene

-   -   A 2a    -   2 2b    -   B2 8    -   C 10    -   D (Val¹¹⁴) 1    -   F 21    -   G 5    -   H2 14    -   I 117    -   J1 7    -   K 6    -   4a 4a    -   4b 4b    -   WA 16    -   1 (Ala¹¹⁴) 1

See Pestka et al. (1997) “Interferon Standardization and Designations” JInterferon Cytokine Res 17: Supplement 1, S9-S14. IFNα B2 is sometimesalso referred to as IFNα B, and is not to be confused with IFNβ. NaturalIFNα from leukocytes (leukocyte IFN), as well as these recombinant humanIFNα protein subtypes are available from PBL Biomedical Labs,Piscataway, N.J. (interferonsource.com). Natural IFNα is a complexmixture of IFNα subtypes. IFNβ has not been detected in the natural IFNαpreparations used herein. Methods for detecting and quantization ofthese interferons, such as ELISA and RIA are known in the art. SeeStaehelin et al. (1981) Methods in Enzymology 79 (S. Pestka, ed.)Academic Press, NY 589-595; Kelder et al. (1986) Methods in Enzymology119 (S. Pestka, ed.) Academic Press, NY 582-587; Stewart (2003) supra;Bennett, et al. (2003) J. Exp. Med. 197(6):711-723; Baechler, et al.(2003) Proc. Natl. Acad. Sci. USA 100(5):2610-2615.

The term “IFNα-producing cell”, as used herein, refers to a specializedleukocyte that is responsible for IFNα production which is broadlyinduced by double stranded RNA (ds)RNA, viruses, bacteria, protozoa,certain cell lines and unmethylated CpG-DNA. Ronnblom and Alm (2004) J.Exp. Med. 194(12):F59-F63.

The phrase “IFNα related condition or disease”, as used herein, refersto abnormal and deleterious diseases or pre-clinical disease states thathave been linked with elevated levels of IFNα in a patient's serum.Examples of such include, but are not limited to SLE, Graft versus HostDisease (GVHD), type 1 diabetes, AIDS (caused by human immunodeficiencyvirus (HIV)), autoimmune thyroiditis, psoriasis and lupus. Methods fordetermining the level of IFNα are known in the art and described herein.

The term “immune response”, as used herein refers to theantigen-specific responses of lymphocytes to foreign substances. Anysubstance that can elicit an immune response is considered to be“immunogenic” and is referred to as an “immunogen”. All immunogens areantigens, however, not all antigens are immunogenic. An immune responsecan be humoral (via antibody activity) or cell-mediated (via T cellactivation).

The terms “immunological binding,” and “immunological bindingproperties”, as used herein refer to the non-covalent interactions ofthe type which occur between an immunoglobulin molecule and an antigenfor which the immunoglobulin is specific. The strength, or affinity ofimmunological binding interactions can be expressed in terms of thedissociation constant (K_(d)) of the interaction, wherein a smallerK_(d) represents a greater affinity. Immunological binding properties ofselected polypeptides can be quantified using methods well known in theart. One such method entails measuring the rates of antigen-bindingsite/antigen complex formation and dissociation, wherein those ratesdepend on the concentrations of the complex partners, the affinity ofthe interaction, and on geometric parameters that equally influence therate in both directions. Thus, both the “on rate constant” (K_(on)) andthe “off rate constant” (K_(off)) may be determined by calculation ofthe concentrations and the actual rates of association and dissociationas are well known in the art. The ratio of K_(off)/K_(on) enablescancellation of all parameters not related to affinity, and is thusequal to the dissociation constant K_(d). See, e.g., Coligan, et al.,CURRENT PROTOCOLS IN IMMUNOLOGY, Wiley, NY (1999).

The term “isolated”, as used herein, refers to an antibody that has beenidentified and separated and/or recovered from a component of itsnatural environment. Contaminant components of its natural environmentare materials that may interfere with diagnostic or therapeutic uses forthe antibody, and may include enzymes, hormones, and other proteinaceousor nonproteinaceous solutes. In preferred embodiments, the antibody willbe purified (1) to greater than 95% by weight of antibody as determinedby the Lowry method, and most preferably more than 99% by weight, (2) toa degree sufficient to obtain at least 15 residues of N-terminal orinternal amino acid sequence by use of a spinning cup sequenator, or (3)to homogeneity by SDS-PAGE under reducing or nonreducing conditionsusing Coomassie blue or, preferably, silver stain. Isolated antibodyincludes the antibody in situ within recombinant cells since at leastone component of the antibody's natural environment will not be present.Ordinarily, however, isolated antibody may be prepared by at least onepurification step. Monoclonal antibodies and variants and derivativesthereof are considered isolated antibodies.

The term “isotype”, as used herein, refers to the antibody class basedon the amino acid sequence of the constant domain of their heavy chains.There are five major isotypes of immunoglobulins: IgA, IgD, IgE, IgG,and IgM, and several of these can be further divided into subclasses,e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.

“Lupus” refers to several diseases or disorders. “Systemic lupuserythematosus” (SLE) is the form of the disease that can affect manyparts of the body. The symptoms of SLE may be mild or serious, and arereviewed herein. “Discoid lupus erythematosus” is a chronic skindisorder in which a red, raised rash appears on the face, scalp, orelsewhere. The raised areas may become thick and scaly and may causescarring. The rash may last for days or years and may recur. A smallpercentage of people with discoid lupus have or develop SLE later.“Subacute cutaneous lupus erythematosus” refers to skin lesions thatappear on parts of the body exposed to sun. “Drug-induced lupus” is aform of lupus caused by certain medications. Symptoms are similar tothose of SLE (arthritis, rash, fever, and chest pain) and they typicallygo away completely when the drug is stopped. The kidneys and brain arerarely involved. “Neonatal lupus” is a rare disease that can occur innewborn babies of women with SLE, Sjögren's syndrome, or no disease atall, and may be caused by autoantibodies in the mother's blood calledanti-Ro (SSA) and anti-La (SSB). At birth, the babies typically have askin rash, liver problems, and low blood counts.

The term “pharmaceutically acceptable carrier”, as used herein, refersto encompasses any of the standard pharmaceutical carriers, e.g., aphosphate buffered saline solution, water, and emulsions, such as anoil/water or water/oil emulsion, and various types of wetting agents.The compositions may also include stabilizers and preservatives and anyof the above noted carriers with the additional provisio that they beacceptable for use in vivo. For examples of carriers, stabilizers andadjuvants, see Martin REMINGTON'S PHARM. SCI., 18th Ed., Mack Publ. Co.,Easton, Pa. (1995), and in the “PHYSICIAN'S DESK REFERENCE”, 58th ed.,Medical Economics, Montvale, N.J. (2004).

The terms, “selectively neutralizes” and “selectively neutralizing”, asused herein, refer to an isolated and purified antibody (such as, butnot limited to a monoclonal antibody) that neutralizes selectively atleast 40% of a bioactivity of one or more “IFNα” protein subtypes, butdoes not significantly neutralize at least one bioactivity of anotherIFNα protein subtype, wherein the bioactivity is activation of the MxApromoter or antiviral activity. Since the different subtypes of IFNαvary in function, it is advantageous to selectively neutralize specificforms of IFNα to control specific functions. The one or more antibodiesof the present invention are specific for IFNα, but are also “selective”for one or more subtypes and not others. In order to selectivelyneutralize one or more IFNα protein subtypes, e.g., A, 2, B2, C, F, G,H2, I, J1, K, 4a, 4b, one or more antigenic epitopes on IFNα that do notsignificantly cross-react with antigenic epitopes on, e.g., the Dsubtype, have been identified, isolated, characterized and purified, asdisclosed herein.

The phrase “does not significantly neutralize”, as used herein, refersto an antibody that neutralizes less than 40% of the bioactivity of aspecified IFNα subtype (or of IFNβ), wherein the bioactivity is measuredby the MxA reporter gene assay (RG assay) or cytopathic effect assay(CPE assay) in accordance with the conditions described herein. In someembodiments, an antibody of the invention does not significantlyneutralize more than 35%, 30%, 25%, 20%, 15%, 10%, 5%, 2% or even notmore than 1% of the bioactivity of the specified IFNα subtype.

The term “subject”, as used herein, refers to a vertebrate, preferably amammal, more preferably a human. Mammals include, but are not limitedto, rodents, simians, humans, farm animals, horses, dogs and cats.

Current SLE treatments are symptomatic and induce globalimmunosuppression; these include glucocorticoids, cyclophosphamide,azathioprine and mycophenolate mofetil. Their efficacy is only partial,and undesirable side-effects such as increased susceptibility toinfections are common. Specifically neutralizing IFN-α in SLE patientsis thus an attractive concept for controlling the disease pathology.Since massively unregulated production of IFN-α by pDCs plays asignificant role in perpetuating the disease cycle, neutralizing IFN-αbioactivity using specific MAb blockade provides a targeted therapeuticagent that does not compromise the ability of patients to mounteffective immune responses to pathogens. A desirable MAb candidateagainst IFN-α would require specific characteristics, including: (i)ability to react against most or all of the human IFN-α subtypesinvolved in the etiology of SLE; (ii) ability to block the biologicalactivities of such IFN-α subtypes, (iii) inability to block either IFN-βor the IFNAR; and/or (iv) high affinity. Neutralizing IFN-α rather thanthe IFNAR may also provide a safer and more specific therapeutic sincethis approach would not affect the antiviral effects of the IFN-βsignaling pathway, which uses the same receptor as IFN-α. To this end,the invention also provides a series of monoclonal antibodies (MAbs)capable of neutralizing human IFN-α. For example, two of theseanti-IFN-α MAbs are capable of blocking the bioactivity of thirteenrecombinant IFN-α subtypes as well as two complex mixtures of IFNproduced upon viral infection. In one aspect, the invention providesseven MAbs that variably neutralize human IFN-α, of which threesignificantly neutralize up to thirteen recombinant IFN-α subtypes andcomplex IFN-α mixtures (both commercially-available leukocyte IFN andsupernatants produced upon infection of PBMCs with flu virus). Two ofthe MAbs, ACO-1 and ACO-2, also consistently block the bioactivity ofserum from SLE patients that exhibit IFN-α signatures by microarrayanalysis. Since ACO-1 and ACO-2 do not significantly neutralize thebioactivity of IFNα protein subytpes D and 1, but do neutralize the IFNαbioactivity of SLE serum, these subtypes are unlikely to be involvedsignificantly in the etiology of SLE. Accordingly, it is desirable totreat SLE using antibodies, such as humanized or non-antigenic (e.g.,deimmunized) variants of ACO-1 and ACO-2, which block the bioactivity ofIFNα subtypes associated with SLE, but do not block the bioactivities ofIFN a protein subytpes (D and 1) that are not significantly associatedwith SLE.

The invention also provides an antibody that selectively neutralizes abioactivity of at least two interferon alpha (“IFNα”) protein subtypesselected from the group consisting of protein subtypes A, 2, B2, C, F,G, H2, I, J1, K, 4a, 4b and WA, but does not significantly neutralize atleast one bioactivity of IFNα protein subtype D; wherein the bioactivityis, e.g., activation of the MxA promoter and/or antiviral activity. Inanother aspect, the invention provides a method for treating a diseaseor condition associated with abnormal expression of at least oneinterferon alpha (“IFNα”) protein subtype selected from protein subtypesA, 2, B2, C, F, G, H2, I, J1, K, 4a, 4b and WA without neutralizing IFNαprotein subtype D antiviral activity, in a subject, includingadministering to the subject an effective amount of one or more of theantibodies described herein. Examples of such antibodies include, butare not limited to, the antibodies designated ACO-1, ACO-2, ACO-3,ACO-4, ACO-5, ACO-6 and antibodies that recognize the same oressentially the same IFNα epitope, or antibodies that compete with anantibody that recognizes essentially the same, or the same IFNα epitopeas any of the foregoing antibodies. Preferably, the antibodies aremonoclonal antibodies. The ATCC deposit numbers of hybridoma cell linesthat produce these monoclonal antibodies are listed hereinbelow.Accordingly, the invention further provides hybridoma cell linesexpressing the ACO-1, ACO-2, ACO-3, ACO-4, ACO-5, ACO-6, and ACO-8antibodies. In one aspect, the antibody binds essentially the same IFNαepitope as the anti-IFNα antibody produced by the hybridoma having ATCCAccession No. PTA-7778. In another aspect, the antibody binds to thesame IFNα epitope as the anti-IFNα antibody produced by the hybridomahaving ATCC Accession No. PTA-7778. In a further aspect, the antibodycompetes with an antibody that binds with essentially the same or thesame IFNα epitope as the anti-IFNα antibody produced by the hybridomahaving ATCC Accession No. PTA-7778.

In one aspect, the invention provides an antibody that binds essentiallythe same IFNα epitope as an antibody selected from the group consistingof ACO-1, ACO-2, ACO-3, ACO-4, ACO-5 and ACO-6. In another aspect, theinvention provides an antibody that competes with essentially the sameIFNα epitope as an antibody selected from the group consisting of ACO-1,ACO-2, ACO-3, ACO-4, ACO-5, and ACO-6. In yet another aspect, theinvention provides an antibody that binds the same epitope as anantibody selected from the group consisting of ACO-1, ACO-2, ACO-3,ACO-4, ACO-5, and ACO-6. In another aspect, the invention provides anantibody that competes with an antibody that binds the same IFNα epitopeas an antibody selected from the group consisting of ACO-1, ACO-2,ACO-3, ACO-4, ACO-5, and ACO-6. The invention further provides celllines, e.g., a hybridoma, which expresses such antibodies.

In another embodiment, the invention provides an antibody thatneutralizes a bioactivity of at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13 or 14 IFNα protein subtypes selected from protein subtypes A, 2, B2,C, F, G, H2, I, J1, K, 4a, 4b and WA, but does not neutralize at leastone bioactivity of IFNα protein subtype D; wherein the bioactivity isactivation of the MxA promoter or antiviral activity. The inventionfurther provides hybridoma cell lines expressing such antibodies.

Monoclonal antibodies are provided that do not neutralize at least onebioactivity of IFNα protein subtype 1, wherein the bioactivity isactivation of the MxA promoter and/or antiviral activity.

In another aspect, the invention provides a monoclonal antibody thatselectively neutralizes a bioactivity the IFNα protein subtypes A, 2,B2, C, F, G, H2, I, K, 4a, 4b and WA, but does not significantlyneutralize the bioactivity of IFNα protein subtypes D and 1, wherein thebioactivity is activation of the MxA promoter and/or antiviral activity.Other embodiments include ACO-1 and ACO-2.

In still another aspect, the invention provides a monoclonal antibodythat selectively neutralizes the bioactivity the IFNα protein subtypesA, 2, B2, C, I, K and 4a, but does not significantly neutralize thebioactivity of IFNα protein subtypes D, F, G, 4b and 1; wherein thebioactivity is activation of the MxA promoter and/or antiviral activity.One such embodiment is the ACO-3 antibody made by the ACO-3 cell andderivatives thereof.

In further aspect, the invention provides an antibody that selectivelyneutralizes the bioactivity the IFNα protein subtypes A, 2, B2, and C,but does not significantly neutralize the bioactivity of IFNα proteinsubtypes D, 4b, and 1, wherein the bioactivity is activation of the MxApromoter and/or antiviral activity. One such embodiment is the ACO-4antibody and derivatives thereof made by the ACO-4 cell and derivativesthereof.

In another aspect, the antibody of this invention selectivelyneutralizes a bioactivity IFNα 4a, but does not selectively neutralizeIFNα 4b, wherein the bioactivity is activation of the MxA promoter.Examples of these antibodies are the antibodies designated ACO-3 andACO-4, and derivatives thereof, made by, e.g., the ACO-3 and ACO-4 cellsand derivatives thereof, respectively.

In yet another aspect, the invention provides a monoclonal antibody thatselectively neutralizes the bioactivity the IFNα protein subtypes A, 2,G, I, K, WA and 1, but does not significantly neutralize the bioactivityof IFNα protein subtypes B2 and D, wherein the bioactivity is activationof the MxA promoter and/or antiviral activity. One such embodiment isthe ACO-5 antibody and derivatives thereof, made by, e.g., the ACO-5cell and derivatives thereof.

In an additional embodiment, the invention provides a monoclonalantibody that selectively neutralizes the bioactivity the IFNα proteinsubtypes 2 and C, but does not neutralize the bioactivity of IFNαprotein subtypes A, B2, C, D, F and 1, wherein the bioactivity isactivation of the MxA promoter. An example is the ACO-6 antibody andderivatives thereof made by the ACO-6 cell and derivatives thereof.

In still another aspect, the invention provides a monoclonal antibodythat selectively neutralizes the bioactivity the IFNα protein subtypesA, 2, B2, D, F, I, 4a, 4b, and 1, but does not significantly neutralizethe bioactivity of IFNα protein subtypes C, H2, K and WA, wherein thebioactivity is activation of the MxA promoter. An example is the ACO-8antibody and derivatives thereof made by the ACO-8 cell and derivativesthereof.

In another aspect, the invention provides a monoclonal anti-interferonalpha (“IFNα”) antibody produced by the hybridoma having ATCC AccessionNo. PTA-7778.

In another aspect, the invention provides an antibody that selectivelyneutralizes a bioactivity of at least two interferon alpha (“IFNα”)protein subtypes with a half maximal effective concentration (EC₅₀) ofless than about 350 ng/ml, more preferably less than about 300 ng/ml,for subtypes selected from the group consisting of protein subtypes A,2, B2, C, F, G, H2, I, J1, 4a, 4b and WA and/or less than about 400ng/ml, more preferably less than about 375 ng/ml for subtype K. Halfmaximal effective concentrations may be determined using any methodavailable, preferably using the RG bioassay as described herein.

The antibody molecules of the present invention may have a high bindingaffinity for IFNα protein subtypes (e.g. nanomolar binding). Affinitymay be measured using any suitable method known in the art, includingthe Biacore assay as described in the examples herein. Preferablyaffinity is measured using recombinant IFNα-A as described in theexamples herein. An antibody molecule according to the present inventionmay have a binding affinity for IFNα-A of less than about 5×10⁻⁹ M, oran antibody molecule according to the present invention may have abinding affinity for IFNα-A of less than 1.5×10⁻⁹ M. Accordingly, in oneembodiment, an antibody molecule of the present invention has a bindingaffinity of between about 9×10⁻⁹ M and about 4×10⁻¹⁰ M. In anotherembodiment, an antibody molecule of the present invention may have abinding affinity of between about 5×10⁻⁹ M and about 1.5×10⁻⁹ M.

The monoclonal antibodies of the invention also include a humanizedantibody, a human antibody, a chimeric antibody, an antibody fragment,such as an Fab fragment, an F(ab′)2 fragment, an Fab′ fragment or anyother fragment(s) known to the skilled artisan. In one example, theantibody is a humanized chimeric antibody.

In yet another embodiment, the invention provides a method of producinga hybridoma cell line by, for example, immunizing a mammal with acomposition including recombinant IFNα subtypes A, B2 and F; fusingsplenocytes from the mammal to a myeloma cell line to producehybridomas; and identifying a hybridoma cell line that produces amonoclonal antibody that selectively neutralizes one or more IFNαprotein subtypes selected from the group consisting of 2, C, G, I, J1,K, 4a, 4b and WA and 1, but does not selectively neutralize IFNα proteinsubtype D.

The term “neutralizes”, as used herein, refers to the ability of anantibody to inhibit one or more biological activities of an IFNα proteinsubtype by at least 40% as measured by the RG, CPE or monocytedifferentiation assays defined herein. For example, the antibodyneutralizes at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%,99% or 100% of a biological activity of an IFNα protein subtype. IFNαbiological activities include transcriptional activation of the MxApromoter (see, e.g., “RG” assay, infra), antiviral activity (e.g.,cytopathic effect assay (“CPE”), and the ability of SLE serum to causedifferentiation of monocytes into dendritic cells. Methods fordetermining the % neutralization using RG assay and the CPE assay aredescribed herein.

The phrases “does not neutralize” or “does not significantlyneutralize”, as used herein, refers to an antibody neutralizes less than40% of a biological activity of an IFNα protein subtype, wherein theneutralizing effect of added antibody as measured by the RG, CPE ormonocyte differentiation assay. For example, the antibody neutralizesless than 35%, or less than 35%, or less than 30%, or less than 25%, orless than 20%, or less than 15%, or less than 10%, or less than 8%, orless than 5%, or less than 3%, or even less than 1% of the bioactivity.

The antibody of the invention may be antibody variants, derivatives orfragments. In one aspect, the antibodies are isolated. In anotheraspect, the antibodies are combined with a suitable carrier. Theantibodies can be isolated from any species, mouse, rat, simian, orrecombinantly produced. Examples of mouse monoclonal antibodies are theantibodies designated ACO-1, ACO-2, ACO-3, ACO-4, ACO-5, ACO-6 andACO-8. Also provided by this invention are the hybridoma cell lines thatproduce these monoclonal antibodies, alone in combination with a carrieror in culture.

Also provided by this invention are polypeptides that include anantibody, variant, derivative or fragment thereof, including but notlimited to immunoglobulin chains and CDRs. The polypeptides preferablybind, inhibit and/or neutralize IFNα as described above, with the sameor similar affinity and/or ability.

The present invention further provides an anti-idiotypic antibodyreactive with any of the antibodies ACO-1 through ACO-6 or ACO-8. Ananti-idiotype antibody is an antibody made against the uniquedeterminants of a single antibody. Anti-idiotype antibodies are usefulfor detecting bound antibodies in immunoassays and other applications.An anti-idiotype antibody of the invention can include or be derivedfrom any mammal, such as but not limited to a human, a mouse, a rabbit,a rat, a rodent, a primate, and the like.

One or more of the above antibodies can be further combined with acarrier, a pharmaceutically acceptable carrier or medical device that issuitable for use of the antibody or related composition in diagnostic ortherapeutic methods. The carrier can be a liquid phase carrier or solidphase carrier, e.g., bead, gel or carrier molecule such as a liposome.The composition can optionally further include at least one furthercompound, protein or composition. An additional example of “carriers”includes therapeutically active agents such as another peptide orprotein (e.g., an Fab′ fragment, a J-chain, another antibody, a toxinand the like). For example, an anti-IFNα antibody of this invention,variant, derivative or fragment thereof can be functionally linked(e.g., by chemical coupling, genetic fusion, noncovalent association orotherwise) to one or more other molecular entities, such as anotherantibody (e.g., to produce a bispecific or a multispecific antibody), acytotoxin, a cellular ligand or an antigen. Accordingly, this inventionencompasses a large variety of antibody conjugates, bi- andmultispecific molecules, and fusion proteins, whether or not they targetthe same epitope as the antibodies of this invention.

Additional examples of carriers are organic molecules (also termedmodifying agents) or activating agents that may be attached covalently,directly or indirectly, to an antibody of this invention. Attachment ofthe molecule can improve pharmacokinetic properties (e.g., increased invivo serum half-life). Examples of organic molecules include, but arenot limited to a hydrophilic polymeric group, a fatty acid group or afatty acid ester group. The term “fatty acid”, as used herein, refersto, e.g., mono-carboxylic acids and di-carboxylic acids. The term“hydrophilic polymeric group”, as used herein, refers to an organicpolymer that is, e.g., more soluble in water than in octane.

Hydrophilic polymers suitable for modifying antibodies of the inventioncan be linear or branched and include, for example, polyalkane glycols(e.g., polyethylene glycol (PEG), monomethoxy-polyethylene glycol(mPEG), polypropylene glycol (PPG) and the like), carbohydrates (e.g.,dextran, cellulose, oligosaccharides, polysaccharides and the like),polymers of hydrophilic amino acids (e.g., polylysine, polyarginine,polyaspartate and the like), polyalkane oxides (e.g., polyethyleneoxide, polypropylene oxide and the like) and polyvinyl pyrolidone. Asuitable hydrophilic polymer for use with the antibody of the inventionmay have a molecular weight of, e.g., about 800 to about 150,000 Daltonsas a separate molecular entity. The hydrophilic polymeric group can besubstituted with one to about six alkyl, fatty acid or fatty acid estergroups. Hydrophilic polymers that are substituted with a fatty acid orfatty acid ester group can be prepared by employing suitable methods.For example, a polymer including an amine group can be coupled to acarboxylate of the fatty acid or fatty acid ester, and an activatedcarboxylate (e.g., activated with N,N-carbonyl diimidazole) on a fattyacid or fatty acid ester can be coupled to a hydroxyl group on apolymer.

Fatty acids and fatty acid esters suitable for modifying antibodies ofthe invention can be saturated or can contain one or more units ofunsaturation. Examples of such include, but are not limited to,n-dodecanoate, n-tetradecanoate, n-octadecanoate, n-eicosanoate,n-docosanoate, n-triacontanoate, n-tetracontanoate,cis-δ-9-octadecanoate, all cis-δ-5,8,11,14-eicosatetraenoate,octanedioic acid, tetradecanedioic acid, octadecanedioic acid,docosanedioic acid, and the like. Suitable fatty acid esters includemono-esters of dicarboxylic acids that include a linear or branchedlower alkyl group. The lower alkyl group can include from one to abouttwelve, preferably one to about six, carbon atoms.

In yet another aspect, the present invention provides a transgenicnonhuman animal, such as a transgenic mouse (also referred to herein asa “Human MAb mouse”), which expresses a fully human monoclonal antibodythat neutralizes at least one IFNα protein subtype similar to anantibody of this invention as defined above. In a particular embodiment,the transgenic nonhuman animal is a transgenic mouse having a genomeincluding a human heavy chain transgene and a human light chaintransgene encoding all or a portion of an anti-alpha V antibody of theinvention. To generate human antibodies, the transgenic nonhuman animalcan be immunized with a purified or enriched preparation of IFNα proteinsubtypes A, B and F. An example of a transgenic nonhuman animal may be,e.g., a transgenic mouse that is capable of producing multiple isotypesof human monoclonal antibodies to IFNα (e.g., IgG, IgA and/or IgM) byundergoing V-D-J recombination and isotype switching. Isotype switchingmay occur by, e.g., classical or non-classical isotype switching.

Accordingly, in another embodiment, the invention provides isolatedcells derived or isolated from a transgenic nonhuman animal as describedabove, e.g., a transgenic mouse, which express human antibodies. Theisolated B-cells can then be immortalized by fusion to an immortalizedcell to provide a source (e.g., a hybridoma) of human antibodies. Thesehybridomas are also included within the scope of the invention.

The present invention further provides at least one antibody method orcomposition, for diagnosing at least one IFNα related condition in acell, tissue, organ, animal or patient and/or, prior to, subsequent to,or during a related condition, as known in the art and/or as describedherein. They are also used to prognose or monitor disease progression.

Also provided is a composition comprising at least one anti-IFNαantibody of this invention, variant, derivative or fragment thereof,suitable for administration in an effective amount to modulate orameliorate IFNα-associated symptoms or treat at least one IFNα relatedcondition in a cell, tissue, organ, animal or patient and/or, prior to,subsequent to, or during a related condition, as known in the art and/oras described herein. The compositions include, for example,pharmaceutical and diagnostic compositions/kits, including apharmaceutically acceptable carrier and at least one anti-IFNα antibodyof this invention, variant, derivative or fragment thereof. As notedabove, the composition can further include additional antibodies ortherapeutic agents which in combination, provide multiple therapiestailored to provide the maximum therapeutic benefit.

Alternatively, a composition of this invention can be co-administeredwith other therapeutic and cytotoxic agents, whether or not linked tothem or administered in the same dosing. They can be coadministeredsimultaneously with such agents (e.g., in a single composition orseparately) or can be administered before or after administration ofsuch agents. Such agents can include corticosteroids, nonsteroidalimmune suppressants, antimalarials, and nonsteroidal anti-inflammatorydrugs. The compositions can be combined with alternative therapies suchas administration of corticosteroids, nonsteroidal immune suppressants,antimalarials, and nonsteroidal anti-inflammatory drugs.

In another aspect, the invention provides methods for selectivelyneutralizing a bioactivity of at least two interferon alpha (“IFNα”)protein subtypes selected from the group consisting of protein subtypesA, 2, B2, C, F, G, H2, I, J1, K, 4a, 4b and WA, without selectivelyneutralizing at least one bioactivity of IFNα protein subtype D; whereinthe bioactivity is activation of the MxA promoter or antiviral activity.The method requires contacting the sample suspected of containing thesubtype with a monoclonal antibody that selectively neutralizes thesubtype. Examples of such antibodies include, but are not limited to theantibodies designated ACO-1, ACO-2, ACO-3, ACO-4, ACO-5 and ACO-6.

Various therapies are provided by this invention. For example, thisspecification discloses methods for ameliorating the symptoms associatedwith abnormal expression of at least one interferon alpha (“IFNα”)protein subtype selected from the group consisting of protein subtypesA, 2, B2, C, F, G, H2, I, K, 4a, 4b and WA without binding IFNα proteinsubtypes D and 1 in a subject, by administering to the subject aneffective amount of an antibody that selectively neutralizes thesubtype. Examples of such are provided above.

The invention provides methods for treating a disease or condition whichresults in abnormal expression of at least one interferon alpha (“IFNα”)protein subtype selected from the group consisting of protein subtypesA, 2, B2, C, F, G, H2, I, K, 4a, 4b and WA without binding IFNα proteinsubtypes D and 1 in a subject, by administering to the subject aneffective amount of an antibody that selectively neutralizes thesubtype. Examples of such are provided above. Further provided aremethods of immunization by administering to a mammal a compositionincluding recombinant IFNα subtypes A, B2 and F. In another embodiment,the immunization method includes administering to a mammal a compositionconsisting essentially of IFNα subtypes A, B2 and F, a pharmaceuticallyacceptable carrier, and optionally, one or more adjuvants. In analternative aspect, immunization raises antibodies that neutralize IFNαprotein subtypes A, B2 and F, but not IFNα protein subtypes D and 1. Ina further aspect, the method and composition neutralizes IFNα 4a and notIFNα 4b (e.g., ACO-3).

For use in vivo, the antibodies and compositions of this invention areadministered or delivered to patients (e.g., human subjects) attherapeutically effective dosages to neutralize selected IFNα subtypes.Additionally, administration or delivery of effective amounts ofantibodies or compositions of this invention can be use to treat orameliorate the symptoms of an IFNα related condition such as SLE,diabetes, psoriasis or AIDS, in a subject using any suitable route ofadministration for antibody-based clinical products. Many are known inthe art, such as injection or infusion.

Dosages Forms. A dosage unit for use of the antibodies of the presentinvention may be a single compound or mixtures thereof with othercompounds. The compounds may be mixed together, form ionic or evencovalent bonds. One or more of the antibodies of the present inventionmay be administered in oral, intravenous (bolus or infusion),intraperitoneal, subcutaneous, or intramuscular form, all using dosageforms well known to those of ordinary skill in the pharmaceutical arts.Depending on the particular location or method of delivery, differentdosage forms, e.g., tablets, capsules, pills, powders, granules,elixirs, tinctures, suspensions, syrups, and emulsions may be used toprovide the antibody(ies) of the present invention to a patient in needof therapy that includes the neutralization of certain IFNα subtypes, asdescribed herein. The antibodies are generally hydrophobic but they maybe administered as any one of known salt forms.

Antibodies are typically administered in admixture with suitablepharmaceutical salts, buffers, diluents, extenders, excipients and/orcarriers (collectively referred to herein as a pharmaceuticallyacceptable carrier or carrier materials) selected based on the intendedform of administration and as consistent with conventionalpharmaceutical practices. Depending on the best location foradministration, the antibodies may be formulated to provide, e.g.,maximum and/or consistent dosing for the particular form for oral,rectal, topical, intravenous injection or parenteral administration.While the antibodies, e.g., humanized forms of ACO-1, ACO-2, ACO-3,ACO-4, ACO-5, ACO-6 and ACO-8, may be administered alone, or incombination, in a pharmaceutically acceptable carrier. The carrier maybe solid or liquid, depending on the type and/or location ofadministration selected.

Techniques and compositions for making useful dosage forms using thepresent invention are described in one or more of the followingreferences: Ansel, Introduction to Pharmaceutical Dosage Forms 2ndEdition (1976); Remington's Pharmaceutical Sciences, 17th ed. (MackPublishing Company, Easton, Pa., 1985); Advances in PharmaceuticalSciences (David Ganderton, Trevor Jones, Eds., 1992); Advances inPharmaceutical Sciences Vol 7. (David Ganderton, Trevor Jones, JamesMcGinity, Eds., 1995); Aqueous Polymeric Coatings for PharmaceuticalDosage Forms (Drugs and the Pharmaceutical Sciences, Series 36 (JamesMcGinity, Ed., 1989); Pharmaceutical Particulate Carriers: TherapeuticApplications: Drugs and the Pharmaceutical Sciences, Vol 61 (AlainRolland, Ed., 1993); Drug Delivery to the Gastrointestinal Tract (EllisHorwood Books in the Biological Sciences. Series in PharmaceuticalTechnology; J. G. Hardy, S. S. Davis, Clive G. Wilson, Eds.); ModernPharmaceutics Drugs and the Pharmaceutical Sciences, Vol 40 (Gilbert S.Banker, Christopher T. Rhodes, Eds.), and the like, relevant portionsincorporated herein by reference.

The antibodies of the present invention may be administered in the formof liposome delivery systems, e.g., small unilamellar vesicles, largeunilamallar vesicles, and multilamellar vesicles, whether charged oruncharged. Liposomes may include one or more: phospholipids (e.g.,cholesterol), stearylamine and/or phosphatidylcholines, mixturesthereof, and the like.

The antibodies may also be coupled to one or more soluble,biodegradable, bioacceptable polymers as drug carriers or as a prodrug.Such polymers may include: polyvinylpyrrolidone, pyran copolymer,polyhydroxylpropylmethacrylamide-phenol,polyhydroxyethylasparta-midephenol, or polyethyleneoxide-polylysinesubstituted with palmitoyl residues, mixtures thereof, and the like.Furthermore, the antibodies may be coupled one or more biodegradablepolymers to achieve controlled release of the antibodies, biodegradablepolymers for use with the present invention include: polylactic acid,polyglycolic acid, copolymers of polylactic and polyglycolic acid,polyepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters,polyacetals, polydihydropyrans, polycyanoacylates, and crosslinked oramphipathic block copolymers of hydrogels, mixtures thereof, and thelike.

For direct delivery to the nasal passages, sinuses, mouth, throat,esophagous, tachea, lungs and alveoli, the antibodies may also bedelivered as an intranasal form via use of a suitable intranasalvehicle. For dermal and transdermal delivery, the antibodies may bedelivered using lotions, creams, oils, elixirs, serums, transdermal skinpatches and the like, as are well known to those of ordinary skill inthat art. Parenteral and intravenous forms may also includepharmaceutically acceptable salts and/or minerals and other materials tomake them compatible with the type of injection or delivery systemchosen, e.g., a buffered, isotonic solution. Examples of usefulpharmaceutical dosage forms for administration of antibodies may includethe following forms.

Injectable solution. A parenteral composition suitable foradministration by injection is prepared by stirring 1.5% by weight ofactive ingredient in deionized water and mixed with, e.g., up to 10% byvolume propylene glycol and water. The solution is made isotonic withsodium chloride and sterilized using, e.g., ultrafiltration. Sterileinjectable solutions are prepared by incorporating the active compoundsin the required amount in the appropriate solvent with various of theother ingredients enumerated above, as required, followed by filteredsterilization. Generally, dispersions are prepared by incorporating thevarious sterilized active ingredients into a sterile vehicle that has abasic dispersion medium. In the case of sterile powders for thepreparation of sterile injectable solutions, useful methods for thepreparation of a dry-powder include, vacuum-drying, spray-freezing,vacuum drying in the presence of heat, and freeze-drying techniques,which yield a powder of the active ingredient plus any additionaldesired ingredient from a previously sterile-filtered solution thereof.The antibodies of the present invention may be delivered as micro ornanoparticles in an injectable form or via pulmonary or other delivery.

Suspensions. In one embodiment, an aqueous suspension may be preparedfor administration so that each 5 ml has, e.g., 0.001-1,000 mg of finelydivided active ingredient, 200 mg of sodium carboxymethyl cellulose, 5mg of sodium benzoate, 1.0 g of sorbitol solution, and saline to 0.01,0.1, 1, 5 or 10 ml.

The effective dose of antibody may include amounts yielding uponreconstitution, if in a wet/dry system, concentrations from about 1.0μg/ml to about 1000 mg/ml, although lower and higher concentrations areoperable and are dependent on the intended delivery vehicle, e.g.,solution formulations will differ from transdermal patch, pulmonary,transmucosal, or osmotic or micro pump methods.

Formulations including an antibody of this invention are providedherein. The formulations of the present invention can be prepared by aprocess that includes mixing at least one antibody of this invention anda preservative selected from the group consisting of phenol, m-cresol,p-cresol, o-cresol, chlorocresol, benzyl alcohol, alkylparaben, (methyl,ethyl, propyl, butyl and the like), benzalkonium chloride, benzethoniumchloride, sodium dehydroacetate and thimerosal or mixtures thereof in anaqueous diluent. Mixing of the antibody and preservative in an aqueousdiluent is carried out using conventional dissolution and mixingprocedures. For example, a measured amount of at least one antibody inbuffered solution is combined with the desired preservative in abuffered solution in quantities sufficient to provide the antibody andpreservative at the desired concentrations. Variations of this processwould be recognized by one of ordinary skill in the art, e.g., the orderthe components are added, whether additional additives are used, thetemperature and pH at which the formulation is prepared, are all factorsthat can be optimized for the concentration and method of administrationused.

The formulation may include one or more preservative or stabilizer suchas phenol, m-cresol, p-cresol, o-cresol, chlorocresol, benzyl alcohol,phenylmercuric nitrite, phenoxyethanol, formaldehyde, chlorobutanol,magnesium chloride (e.g., hexahydrate), alkylparaben (methyl, ethyl,propyl, butyl and the like), benzalkonium chloride, benzethoniumchloride, sodium dehydroacetate and thimerosal, or mixtures thereof inan aqueous diluent. Any suitable concentration or mixture can be used asknown in the art, such as 0.001-5%, or any range or value therein, suchas, but not limited to 0.001, 0.003, 0.005, 0.009, 0.01, 0.02, 0.03,0.05, 0.09, 0.1, 0.2, 0.3, 0.4., 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2,1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6,2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0,4.3, 4.5, 4.6, 4.7, 4.8, 4.9, or any range or value therein.Non-limiting examples include, no preservative(s), or preservatives suchas, e.g., 0.1-2% m-cresol (e.g., 0.2, 0.3. 0.4, 0.5, 0.9, 1.0%), 0.1-3%benzyl alcohol (e.g., 0.5, 0.9, 1.1., 1.5, 1.9, 2.0, 2.5%), 0.001-0.5%thimerosal (e.g., 0.005, 0.01), 0.001-2.0% phenol (e.g., 0.05, 0.25,0.28, 0.5, 0.9, 1.0%), 0.0005-1.0% alkylparaben(s) (e.g., 0.00075,0.0009, 0.001, 0.002, 0.005, 0.0075, 0.009, 0.01, 0.02, 0.05, 0.075,0.09, 0.1, 0.2, 0.3, 0.5, 0.75, 0.9, and 1.0%).

The compositions and formulations can be provided to patients as clearsolutions or as dual vials including a vial of lyophilized antibody thatis reconstituted with a second vial containing the aqueous diluent.Either a single solution vial or dual vial requiring reconstitution canbe reused multiple times and can suffice for a single or multiple cyclesof patient treatment and thus provides a more convenient treatmentregimen than currently available. Recognized devices including thesesingle vial systems include those pen-injector devices for delivery of asolution such as BD Pens, BD Autojectore, Humaject™ NovoPen™, B-DTMPen,AutoPen™, and OptiPen™, GenotropinPen™, Genotronorm Pen™, Humatro Pen™,Reco-Pen™, Roferon Pen™, Biojector™, Iject™, J-tip Needle-FreeInjector™, Intraject™, Medi-Ject, e.g., as made or developed by BectonDickensen (Franklin Lakes, N.J. available at bectondickenson.com),Disetronic (Burgdorf, Switzerland, available at disetronic.com; Bioject,Portland, Oreg. (available at bioject.com); National Medical Products,Weston Medical (Peterborough, UK, available at weston-medical.com),Medi-Ject Corp (Minneapolis, Minn., available at mediject.com).

The invention provides an article of manufacture, including packagingmaterial and at least one vial including a solution of at least antibodyas of this invention with the prescribed buffers and/or preservatives,optionally in an aqueous diluent, wherein the packaging materialincludes a label that indicates that such solution can be held over aperiod of 1, 2, 3, 4, 5, 6, 9, 12, 18, 20, 24, 30, 36, 40, 48, 54, 60,66, 72 hours or greater. The invention further includes an article ofmanufacture, including packaging material, a first vial including atleast one lyophilized antibody of this invention and a second vialincluding an aqueous diluent of prescribed buffer or preservative,wherein the packaging material includes a label that instructs a patientto reconstitute the antibody in the aqueous diluent to form a solutionthat can be held over a period of twenty-four hours or greater.

Kits. The present invention also includes pharmaceutical kits useful,for example, for the treatment of a disease conditions, the kit mayinclude one or more containers that include the pharmaceuticalcomposition that may be provided as is, diluted or resuspended into atherapeutically effective amount of antibodies. Such kits may furtherinclude, if desired, one or more of various conventional pharmaceuticalkit components, such as, for example, containers with one or morepharmaceutically acceptable carriers, liquids, additional containers,etc., as will be readily apparent to those skilled in the art. Printedinstructions, either as inserts or as labels, indicating quantities ofthe components to be administered, guidelines for administration, and/orguidelines for mixing the components, may also be included in the kit.It should be understood that although the specified materials andconditions are important in practicing the invention, unspecifiedmaterials and conditions are not excluded so long as they do not preventthe benefits of the invention from being realized.

Antibodies. The antibodies of this invention include monoclonalantibodies. They also can be IFNα-neutralizing functional fragments,antibody derivatives or antibody variants. They can be chimeric,humanized, or totally human. A functional fragment of an antibodyincludes, but is not limited to, Fab, Fab′, Fab₂, Fab′₂, and singlechain variable regions. Antibodies can be produced in cell culture,e.g., in bacteria, yeast, plants or plant cells, insects or insectcells, eukaryotic cell, or in various animals, including but not limitedto cows, rabbits, goats, mice, rats, hamsters, guinea pigs, sheep, dogs,cats, monkeys, chimpanzees, apes, etc. So long as the fragment orderivative retains specificity of binding or neutralization ability asthe antibodies of this invention it can be used. Antibodies can betested for specificity of binding by comparing binding to appropriateantigen to binding to irrelevant antigen or antigen mixture under agiven set of conditions. If the antibody binds to the appropriateantigen at least 2, 5, 7, and even 10 times more than to irrelevantantigen or antigen mixture then it is considered to be specific.Specific assays, e.g., ELISA, for determining specificity are describedinfra.

The antibodies also are characterized by their ability to neutralize oneor more biological activity of an IFNα protein subtype, such as, but notlimited to, transcriptional activation of the MxA promoter or of anotherpromoter that is inducible by IFNα, antiviral activity, ability of SLEserum to cause differentiation of monocytes into dendritic cells.

The monoclonal antibodies of the invention can be generated usingconventional hybridoma techniques known in the art and well-described inthe literature. For example, a hybridoma is produced by fusing asuitable immortal cell line (e.g., a myeloma cell line such as, but notlimited to, Sp2/0, Sp2/0-AG14, NSO, NS1, NS2, AE-1, L.5, >243,P3X63Ag8.653, Sp2 SA3, Sp2 MAI, Sp2 SS1, Sp2 SA5, U397, MLA 144, ACT IV,MOLT4, DA-1, JURKAT, WEHI, K-562, COS, RAJI, NIH 3T3, HL-60, MLA 144,NAMAIWA, NEURO 2A, CHO, PerC.6, YB2/O) or the like, or heteromyelomas,fusion products thereof, or any cell or fusion cell derived therefrom,or any other suitable cell line as known in the art (see, e.g.,www.atcc.org, www.lifetech.com., and the like), with antibody producingcells, such as, but not limited to, isolated or cloned spleen,peripheral blood, lymph, tonsil, or other immune or B cell containingcells, or any other cells expressing heavy or light chain constant orvariable or framework or CDR sequences, either as endogenous orheterologous nucleic acid, as recombinant or endogenous, viral,bacterial, algal, prokaryotic, amphibian, insect, reptilian, fish,mammalian, rodent, equine, ovine, goat, sheep, primate, eukaryotic,genomic DNA, cDNA, rDNA, mitochondrial DNA or RNA, chloroplast DNA orRNA, hnRNA, mRNA, tRNA, single, double or triple stranded, hybridized,and the like or any combination thereof. Antibody producing cells canalso be obtained from the peripheral blood or, preferably the spleen orlymph nodes, of humans or other suitable animals that have beenimmunized with the antigen of interest. Any other suitable host cell canalso be used for expressing-heterologous or endogenous nucleic acidencoding an antibody, specified fragment or variant thereof, of thepresent invention. The fused cells (hybridomas) or recombinant cells canbe isolated using selective culture conditions or other suitable knownmethods, and cloned by limiting dilution or cell sorting, or other knownmethods.

Other suitable methods of producing or isolating antibodies of therequisite specificity can be used, including, but not limited to,methods that select recombinant antibody from a peptide or proteinlibrary (e.g., but not limited to, a bacteriophage, ribosome,oligonucleotide, RNA, cDNA, or the like, display library; e.g., asavailable from various commercial vendors such as Cambridge AntibodyTechnologies (Cambridgeshire, UK), MorphoSys (Martinsreid/Planegg,Del.), Biovation (Aberdeen, Scotland, UK), Bioinvent (Lund, Sweden), andAntitope (Cambridge, UK) using methods known in the art. See U.S. Pat.Nos. 4,704,692; 5,723,323; 5,763,192; 5,814,476; 5,817,483; 5,824,514;5,976,862. Alternative methods rely upon immunization of transgenicanimals (e.g., SCID mice, Nguyen et al. (1977) Microbiol. Immunol.41:901-907 (1997); Sandhu et al. (1996) Crit. Rev. Biotechnol.16:95-118; Eren et al. (1998) Immunol. 93:154-161 that are capable ofproducing a repertoire of human antibodies, as known in the art and/oras described herein. Such techniques, include, but are not limited to,ribosome display (Hanes et al. (1997) Proc. Natl. Acad. Sci. USA,94:4937-4942; Hanes et al., (1998) Proc. Natl. Acad. Sci. USA,95:14130-14135); single cell antibody producing technologies (e.g.,selected lymphocyte antibody method (“SLAM”) (U.S. Pat. No. 5,627,052,Wen et al. (1987) J. Immunol. 17:887-892; Babcook et al., Proc. Natl.Acad. Sci. USA (1996) 93:7843-7848); gel microdroplet and flow cytometry(Powell et al. (1990) Biotechnol. 8:333-337; One Cell Systems,(Cambridge, Mass.); Gray et al. (1995) J. Imm. Meth. 182:155-163; Kennyet al. (1995) Bio/Technol. 13:787-790); B-cell selection (Steenbakkerset al. (1994) Molec. Biol. Reports 19:125-134 (1994).

Antibody variants of the present invention can also be prepared usingdelivering a polynucleotide encoding an antibody of this invention to asuitable host such as to provide transgenic animals or mammals, such asgoats, cows, horses, sheep, and the like, that produce such antibodiesin their milk. These methods are known in the art and are described forexample in U.S. Pat. Nos. 5,827,690; 5,849,992; 4,873,316; 5,849,992;5,994,616; 5,565,362; and 5,304,489.

The term “antibody variant”, as used herein, refers topost-translational modification to linear polypeptide sequence of theantibody or fragment. For example, U.S. Pat. No. 6,602,684 B1 describesa method for the generation of modified glycol-forms of antibodies,including whole antibody molecules, antibody fragments, or fusionproteins that include a region equivalent to the Fc region of animmunoglobulin, having enhanced Fc-mediated cellular toxicity, andglycoproteins so generated.

Antibody variants also can be prepared by delivering a polynucleotide ofthis invention to provide transgenic plants and cultured plant cells(e.g., but not limited to tobacco, maize, and duckweed) that producesuch antibodies, specified portions or variants in the plant parts or incells cultured therefrom. For example, Cramer et al. (1999) Curr. Top.Microbiol. Immunol. 240:95-118 and references cited therein, describethe production of transgenic tobacco leaves expressing large amounts ofrecombinant proteins, e.g., using an inducible promoter. Transgenicmaize have been used to express mammalian proteins at commercialproduction levels, with biological activities equivalent to thoseproduced in other recombinant systems or purified from natural sources.See, e.g., Hood et al., Adv. Exp. Med. Biol. (1999) 464:127-147 andreferences cited therein. Antibody variants have also been produced inlarge amounts from transgenic plant seeds including antibody fragments,such as single chain antibodies (scFv's), including tobacco seeds andpotato tubers. See, e.g., Conrad et al. (1998) Plant Mol. Biol.38:101-109 and reference cited therein. Thus, antibodies of the presentinvention can also be produced using transgenic plants, according toknow methods.

Antibody derivatives can be produced, for example, by adding exogenoussequences to modify immunogenicity or reduce, enhance or modify binding,affinity, on-rate, off-rate, avidity, specificity, half-life, or anyother suitable characteristic. Generally part or all of the non-human orhuman CDR sequences are maintained while the non-human sequences of thevariable and constant regions are replaced with human or other aminoacids. In general, the CDR residues are directly and most substantiallyinvolved in influencing antigen binding.

Monoclonal antibodies produced in mice (or in other non-human animals)carry the risk in therapy that humans can develop antibodies to theanimal MAb. The human antibodies can then reduce the effectiveness ofthe animal Mab and can also result in an allergic reaction. This problemcan be avoided by constructing antibodies that are not recognized asforeign. Methods for constructing such antibodies are know in the art,and are often based on grafting the CDR region of the animal MAb onto animmunoglobulin backbone of a target host. The most common method ishumanization, which can be accomplished by grafting the CDRs of ananimal antibody onto the framework of a human immunoglobulin. In somecases, a few amino acid residues from framework of the animal antibodyare retained to preserve the integrity of the antigen binding site.

Humanization or engineering of antibodies of the present invention canbe performed using any known method, such as but not limited to thosedescribed in U.S. Pat. Nos. 5,723,323, 5,976,862, 5,824,514, 5,817,483,5,814,476, 5,763,192, 5,723,323, 5,766,886, 5,714,352, 6,204,023,6,180,370, 5,693,762, 5,530,101, 5,585,089, 5,225,539; and 4,816,567.Methods for antibody humanization based on computer modeling andvariable regions are known to those of skill in the art. See, forexample, Tsurushita et al. (2005) Humanized Antibodies and theirApplications 36(1):69-83; the contents of which are incorporated byreference.

In one humanization method, rather than grafting the entire animal CDRsonto a human framework, only the specificity determining residues (SDRs)(the most critical residues of the CDR for the antibody-ligand binding)are grafted onto the human framework (Kashmiri et al. (2005) HumanizedAntibodies and their Applications 36(1):25-34; the contents of which areincorporated by reference). In an alternative approach to humanization,human framework sequences from the set of human germline genes arechosen based on the structural similarity of the human CDR to those ofthe animal CDR to be humanized (Hwang et al. (2005) Humanized Antibodiesand their Applications 36(1):35-42; the contents of which areincorporated by reference).

Framework shuffling is another approach to humanization that allows forthe identification of human framework combinations that will support thefunctional features of mouse or other animal CDRs, without the need forrational design or structural information. In this method, combinatorialFab libraries are created by in-frame fusion of the CDRs of an animalMAb to pools of corresponding human frameworks that include all knownheavy and light chain human germline genes. The Fab libraries may thenbe screened for antigen binding. The light and heavy chains of theparental Mab may be successively humanized in a further selectionprocess. See Dall'Acqua et al. (2005) Humanized Antibodies and theirApplications 36(1):43-60; the contents of which are incorporated byreference.

In an alternative approach, which has been successfully used to make atleast one FDA approved antibody for use in humans, guided selection canbe used to make a serial transition from rodent to human versions ofrodent antibodies through the use of a panel of human antibodies withsimilar characteristics to the starting rodent antibody, and phage orribosome display. See Osbourn et al. (2005) Humanized Antibodies andtheir Applications 36(1):61-68; the contents of which are incorporatedby reference. In this approach, the panel of human antibodies or Vregions are screened for binding of an antigen of interest. Theresulting antibody is entirely of human origin.

Techniques for making partially to fully human antibodies are known inthe art and any such techniques can be used. According to oneembodiment, fully human antibody sequences are made in a transgenicmouse which has been engineered to express human heavy and light chainantibody genes. Multiple strains of such transgenic mice have been madewhich can produce different classes of antibodies. B cells fromtransgenic mice that are producing a desirable antibody can be fused tomake hybridoma cell lines for continuous production of the desiredantibody. (See for example, Russel et al. (2000) Infection and ImmunityApril 2000:1820-1826; Gallo et al. (2000) European J. of Immun.30:534-540; Green (1999) J. of Immun. Methods 231:11-23; Yang et al.(1999) J. of Leukocyte Biology 66:401-410; Yang (1999) Cancer Research59(6):1236-1243; Jakobovits (1998) Advanced Drug Delivery Reviews31:33-42; Green, L. and Jakobovits (1998) J. Exp. Med. 188(3):483-495;Jakobovits (1998) Exp. Opin. Invest. Drugs 7(4):607-614; Tsuda et al.(1997) Genomics 42:413-421; Sherman-Gold (1997) Genetic Engineering News17(14); Mendez et al. (1997) Nature Genetics 15:146-156; Jakobovits(1996) WEIR'S HANDBOOK OF EXPERIMENTAL IMMUNOLOGY, THE INTEGRATED IMMUNESYSTEM Vol. IV, 194.1-194.7; Jakobovits (1995) Current Opinion inBiotechnology 6:561-566; Mendez et al. (1995) Genomics 26:294-307;Jakobovits (1994) Current Biology 4(8):761-763; Arbones et al. (1994)Immunity 1(4):247-260; Jakobovits (1993) Nature 362(6417):255-258;Jakobovits et al. (1993) Proc. Natl. Acad. Sci. USA 90(6):2551-2555;U.S. Pat. No. 6,075,181.)

Human monoclonal antibodies can also be produced by a hybridoma whichincludes a B cell obtained from a transgenic nonhuman animal, e.g., atransgenic mouse, having a genome including a human heavy chaintransgene and a light chain transgene fused to an immortalized cell. Theantibodies of this invention also can be modified to create chimericantibodies. Chimeric antibodies are those in which the various domainsof the antibodies' heavy and light chains are coded for by DNA from morethan one species. See, e.g., U.S. Pat. No. 4,816,567.

Any recombinant antibody or the antibody fragment thereof according tothe present invention may be used, so long as it can react specificallywith at least two interferon alpha (“IFNα”) protein subtypes selectedfrom the group consisting of protein subtypes A, 2, B2, C, F, G, H2, I,J1, K, 4a, 4b and WA, but does not neutralize at least one bioactivityof IFNα protein subtype D. The antibody may also neutralize the IFNα, asmeasured in known bioassays, e.g., the bioactivity is activation of theMxA promoter or antiviral activity. One such antibody is an antibodythat reacts specifically with one or more IFNα subtypes A, 2, B2, C, F,G, H2, I, J1, K, 4a, 4b and WA, but not subtype D, and includes CDRs,derivatives or portions thereof selected from:

V_(H)1 having the amino acid sequence of SEQ ID NO:4;

V_(H)2 having the amino acid sequence of SEQ ID NO:6;

V_(H)3 having the amino acid sequence of SEQ ID NO:8.

V_(L)1 having the amino acid sequence of SEQ ID NO:12;

V_(L)2 having the amino acid sequence of SEQ ID NO:14;

V_(L)3 having the amino acid sequence of SEQ ID NO:16; derivatives andcombinations thereof.

The antibodies may also include antibodies and/or antibody fragments inwhich one or more amino acids are deleted, added, substituted and/orinserted in these amino acid sequences and which specifically react withIFNα subtypes A, 2, B2, C, F, G, H2, I, J1, K, 4a, 4b and WA are alsoincluded within the scope of the present invention.

In the present invention, one or more amino acid deletions,substitutions, insertions or additions in the amino acid sequence refersto modifications and/or mutations of one or more amino acids that aredeleted, substituted, inserted and/or added at one or more positions inthe backbone of an immunoglobulin. The one or more deletions,substitutions, insertions and/or additions may be caused in the sameamino acid sequence simultaneously. Also, the amino acid residuesubstituted, inserted or added can be natural or non-natural. Examplesof the natural amino acid residue include L-alanine, L-asparagine,L-aspartic acid, L-glutanine, L-glutamic acid, glycine, L-histidine,L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine,L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine,L-cysteine, and the like.

Examples of amino acid residues that may be substituted may be foundwithin one or more of the following groups, for example:

Group A: leucine, isoleucine, norleucine, valine, norvaline, alanine,2-aminobutanoic acid, methionine, O-methylserine, t-butylglycine,t-butylalanine, cyclohexylalanine;

Group B: aspartic acid, glutamic acid, isoaspartic acid, isoglutamicacid, 2-aminoadipic acid, 2-aminosuberic acid;

Group C: asparagine, glutamine;

Group D: lysine, arginine, ornithine, 2,4-diaminobutanoic acid,2,3-diaminopropionic acid;

Group E: proline, 3-hydroxyproline, 4-hydroxyproline;

Group F: serine, threonine, homoserine; and

Group G: phenylalanine, tyrosine.

The term “antibody derivative” further includes “linear antibodies”. Theprocedure for making linear antibodies is known in the art and describedin Zapata, et al. (1995) Protein Eng. 8(10):1057-1062. Briefly, linearantibodies include a pair of tandem Fd segments (V_(H)—C_(H)1-VH—C_(H)1) which form a pair of antigen binding regions. Linearantibodies can be bispecific or monospecific.

The antibodies of this invention can be recovered and purified fromrecombinant cell cultures by known methods including, but not limitedto, protein A purification, ammonium sulfate or ethanol precipitation,acid extraction, anion or cation exchange chromatography,phosphocellulose chromatography, hydrophobic interaction chromatography,affinity chromatography, hydroxylapatite chromatography and lectinchromatography. High performance liquid chromatography (“HPLC”) can alsobe used for purification.

Antibodies of the present invention include naturally purified products,products of chemical synthetic procedures, and products produced byrecombinant techniques from a eukaryotic host, including, for example,yeast, higher plant, insect and mammalian cells, or alternatively from aprokaryotic cells as described above.

In some aspects of this invention, it will be useful to detectably ortherapeutically label the antibody. Methods for conjugating antibodiesto these agents are known in the art. For the purpose of illustrationonly, antibodies can be labeled with a detectable moiety such as aradioactive atom, a chromophore, a fluorophore, or the like. Suchlabeled antibodies can be used for diagnostic techniques, either invivo, or in an isolated test sample. Antibodies can also be conjugated,for example, to a pharmaceutical agent, such as chemotherapeutic drug ora toxin. They can be linked to a cytokine, to a ligand, to anotherantibody. Suitable agents for coupling to antibodies to achieve ananti-tumor effect include cytokines, such as interleukin 2 (IL-2) andTumor Necrosis Factor (TNF); photosensitizers, for use in photodynamictherapy, including aluminum (III) phthalocyanine tetrasulfonate,hematoporphyrin, and phthalocyanine; radionucleotides, such asiodine-131(¹¹³I), yttrium-90 (⁹⁰Y), bismuth-212 (²¹²Bi), bismuth-213(²¹³Bi), technetium-99m (^(99m)Tc), rhenium-186 (¹⁸⁶Re), and rhenium-188(¹⁸⁸Re); antibiotics, such as doxorubicin, adriamycin, daunorubicin,methotrexate, daunomycin, neocarzinostatin, and carboplatin; bacterial,plant, and other toxins, such as diphtheria toxin, pseudomonas exotoxinA, staphylococcal enterotoxin A, abrin-A toxin, ricin A (deglycosylatedricin A and native ricin A), TGF-alpha toxin, cytotoxin from Chinesecobra (naja naja atra), and gelonin (a plant toxin); ribosomeinactivating proteins from plants, bacteria and fungi, such asrestrictocin (a ribosome inactivating protein produced by Aspergillusrestrictus), saporin (a ribosome inactivating protein from Saponariaofficinalis), and RNase; tyrosine kinase inhibitors; Iy207702 (adifluorinated purine nucleoside); liposomes containing anti cysticagents (e.g., antisense oligonucleotides, plasmids which encode fortoxins, methotrexate, etc.); and other antibodies or antibody fragments,such as F(ab).

With respect to preparations containing antibodies covalently linked toorganic molecules, they can be prepared using suitable methods, such asby reaction with one or more modifying agents. Examples of such includemodifying and activating groups. A “modifying agent” as the term is usedherein, refers to a suitable organic group (e.g., hydrophilic polymer, afatty acid, a fatty acid ester) that includes an activating group.Specific examples of these are provided supra. An “activating group” isa chemical moiety or functional group that can, under appropriateconditions, react with a second chemical group thereby forming acovalent bond between the modifying agent and the second chemical group.Examples of such are electrophilic groups such as tosylate, mesylate,halo (chloro, bromo, fluoro, iodo), N-hydroxysuccinimidyl esters (NHS),and the like. Activating groups that can react with thiols include, forexample, maleimide, iodoacetyl, acrylolyl, pyridyl disulfides,5-thiol-2-nitrobenzoic acid thiol (TNB-thiol), and the like. An aldehydefunctional group can be coupled to amine- or hydrazide-containingmolecules, and an azide group can react with a trivalent phosphorousgroup to form phosphoramidate or phosphorimide linkages. Suitablemethods to introduce activating groups into molecules are known in theart (see for example, Hermanson (1996) BIOCONJUGATE TECHNIQUES, AcademicPress: San Diego, Calif.). An activating group can be bonded directly tothe organic group (e.g., hydrophilic polymer, fatty acid, fatty acidester), or through a linker moiety, for example a divalent C₁-C₁₂ groupwherein one or more carbon atoms can be replaced by a heteroatom such asoxygen, nitrogen or sulfur. Suitable linker moieties include, forexample, tetraethylene glycol. Modifying agents that include a linkermoiety can be produced, for example, by reacting a mono-Boc-alkyldiamine(e.g., mono-Boc-ethylenediamine, mono-Boc-diaminohexane) with a fattyacid in the presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide(EDC) to form an amide bond between the free amine and the fatty acidcarboxylate. The Boc protecting group can be removed from the product bytreatment with trifluoroacetic acid (TFA) to expose a primary amine thatcan be coupled to another carboxylate as described, or can be reactedwith maleic anhydride and the resulting product cyclized to produce anactivated maleimido derivative of the fatty acid.

The modified antibodies of the invention can be produced by reacting ahuman antibody or antigen-binding fragment with a modifying agent. Forexample, the organic moieties can be bonded to the antibody in anon-site specific manner by employing an amine-reactive modifying agent,for example, an NHS ester of PEG. Modified human antibodies orantigen-binding fragments can also be prepared by reducing disulfidebonds (e.g., intra-chain disulfide bonds) of an antibody orantigen-binding fragment. The reduced antibody or antigen-bindingfragment can then be reacted with a thiol-reactive modifying agent toproduce the modified antibody of the invention. Modified humanantibodies and antigen-binding fragments including an organic moietythat is bonded to specific sites of an antibody of the present inventioncan be prepared using suitable methods, such as reverse proteolysis. Seegenerally, Hermanson (1996) BIOCONJUGATE TECHNIQUES, Academic Press: SanDiego, Calif. (1996).

Preparation and Isolation of Proteins and Polypeptides. Polypeptides andproteins are necessary components of various methods of this invention.For example, recombinant antibodies, variants, derivatives and fragmentsthereof can be obtained by chemical synthesis using a commerciallyavailable automated peptide synthesizer such as those manufactured byPerkin Elmer/Applied Biosystems, Inc., Model 430A or 431A, Foster City,Calif., USA. The synthesized protein or polypeptide can be precipitatedand further purified, for example by high performance liquidchromatography (HPLC). Alternatively, the proteins and polypeptides canbe obtained by known recombinant methods as described herein using thehost cell and vector systems described above. They can also be preparedby enzymatic digestion or cleavage of naturally occurring proteins.

Proteins and peptides can be isolated or purified by standard methodsincluding chromatography (e.g., ion exchange, affinity, and sizingcolumn chromatography), centrifugation, differential solubility, or byany other standard technique for protein purification. Alternatively,affinity tags such as hexa-His (Invitrogen), Maltose binding domain (NewEngland Biolabs), influenza coat sequence (Kolodziej, et al., (1991)Methods Enzymol. 194:508-509), and glutathione-S-transferase can beattached to the peptides of the invention to allow easy purification bypassage over an appropriate affinity column. Isolated peptides can alsobe physically characterized using such techniques as proteolysis,nuclear magnetic resonance, and x-ray crystallography.

It is well known that modifications can be made to any peptide toprovide it with altered properties. Peptides for use in this inventioncan be modified to include unnatural amino acids. Thus, the peptides mayinclude D-amino acids, a combination of D- and L-amino acids, andvarious “designer” amino acids (e.g., β-methyl amino acids, C-β-methylamino acids, and N-α-methyl amino acids, etc.) to convey specialproperties to peptides.

In a further embodiment, subunits of peptides that confer usefulchemical and structural properties will be chosen. For example, peptidesincluding D-amino acids may be resistant to L-amino acid-specificproteases in vivo. Modified compounds with D-amino acids may besynthesized with the amino acids aligned in reverse order to produce thepeptides of the invention as retro-inverso peptides. In addition, thepresent invention envisions preparing peptides that have better definedstructural properties, and the use of peptidomimetics, andpeptidomimetic bonds, such as ester bonds, to prepare peptides withnovel properties. In another embodiment, a peptide may be generated thatincorporates a reduced peptide bond, i.e., R1-CH₂NH—R2, where R1, and R2are amino acid residues or sequences. A reduced peptide bond may beintroduced as a dipeptide subunit. Such a molecule would be resistant topeptide bond hydrolysis, e.g., protease activity. Such molecules wouldprovide peptides with unique function and activity, such as extendedhalf-lives in vivo due to resistance to metabolic breakdown, or proteaseactivity. Furthermore, it is well known that in certain systemsconstrained peptides show enhanced functional activity (Hruby (1982)Life Sciences 31:189-199 and Hruby et al. (1990) Biochem J.268:249-262); the present invention provides a method to produce aconstrained peptide that incorporates random sequences at all otherpositions.

Assays for IFNα Biological Activity. Differentiation of Monocytes. Thegeneration of activated T and B lymphocytes requires the recruitment andmaturation of antigen presenting cells (“APCs”). These APCs include Bcells, monocytes/macrophages and dendritic cells. The serum of SLEpatients contains IFNα which can activate DCs and the activated activitycan blocked with polyclonal or monoclonal antibody preparations. Methodsto detect and quantitate this activity are described in the scientificand patent literature (e.g., see paragraphs 0136 through 0150 of patentpublication number U.S. 2004/0067232 A1), relevant portions incorporatedherein by reference.

Activation of the MxA promoter. The ability of IFNα to activate the MxApromoter, and the ability of the anti-IFNα monoclonal antibodies of theinvention to block this activation can be measured using reporter geneassays where the MxA promoter is fused to a reporter gene, such aschloramphenicol acetyltransferase (CAT) or luciferase (luc), preferablyluciferase. Assays for CAT and luciferase are known to those of skill inthe art. Preferably, the activity of the MxA promoter is measured inA549 cells stably transformed with an MxA promoter/reporter gene fusionconstruct. A549 cells are a lung carcinoma cell line available throughthe ATCC (product number CCl-185). The MxA (a.k.a. Mx1) promoter can behuman, mouse or rat. The sequence and structure of the human MxApromoter is disclosed in Genbank Accession number X55639, Chang et al.(1991) Arch Virol. 117:1-15; and Ronni et al. (1998) J InterferonCytokine Res. 18:773-781. Human MxA promoter/luciferase fusionconstructs and luciferase assays are disclosed in U.S. patentapplication 20040209800 and Rosmorduc et al. (1999) J of Gen Virol80:1253-1262. Human MxA promoter/CAT fusion constructs and CAT assaysare disclosed in Fernandez et al. (2003) J Gen Virol 84:2073-2082 andFray et al. (2001) J Immunol Methods 249:235-244. The mouse MxA (Mx1)promoter is disclosed in Genbank accession number M21104; Hug et al.(1988) Mol Cell Biol 8:3065-3079; and Lleonart et al. (1990)Biotechnology 8:1263-1267. A mouse MxA promoter/luciferase fusionconstruct and a luciferase assay are disclosed in Canosi et al. (1996) JImmunol Methods 199:69-67.

EXAMPLES Materials and Methods

Sources of human IFN-α. Recombinant IFN-α subtype proteins were obtainedfrom PBL Biomedical Laboratories (PBL). The subtypes and specificactivities determined by the manufacturer included: IFN-α A (3.8×10⁸U/mg); IFN-α2 (2.77×10⁸ U/mg); IFN-α B2 (4.63×10⁸ U/mg); IFN-α C(2.31×10⁸ U/mg); IFN-α D (7.5×10⁷ U/mg); IFN-α F (3.6×10⁸ U/mg); IFN-α G(2.33×10⁸ U/mg); IFN-α H2 (1.05×10⁸ U/mg); IFN-α I (1.4×10⁸ U/mg); IFN-αJ1 (2.6×10⁸ U/mg); IFN-α K (1.48×10⁸ U/mg); IFN-α1 (1.4×10⁸ U/mg);IFN-α4a (2.12×10⁸ U/mg); IFN-α4b (1.8×10⁸ U/mg); IFN-α WA (2.4×10⁸U/mg); and IFN-β (8.23×10⁷ U/mg). Leukocyte IFN, was purchased fromSigma (1-2396, Lot #111K1603).

PBMC-flu supernatant (PBMC-flu), which contains a complex mixture ofhuman IFNα subtypes, was prepared in-house by infection of human PBMCsfrom buffy coats with Influenza A/PR/8/34 (H1N1) (Charles RiverLaboratories, Lot #4×PR011022) at a viral titer of 1 HAU/pDC.Specifically, PBMCs (peripheral blood monocytic cells) were harvestedfrom a human buffy coat by centrifuging over Ficoll and collecting thePBMC-containing interface. FACS staining/analysis was performed toconfirm the presence of plasmacytoid DCs and determine their percentagewithin the PBMCs and stained with fluorescence-conjugated antibodiesspecific for Lin, CD3, CD14, CD16, CD19, CD56, CD123, HLA-DR, and CD11c(BD Pharmingen). pDCs were characterized as CD14 negative, CD 11cnegative and CD123 positive. The flu virus stock (Specific Pathogen-FreeAvian Supply; Influenza A/PR/8/34 (H1N1) (Cat. # 490710; Lot #4×PR011022), Final HA titer per 0.05 mL: 1:16,777,216; Charles RiverLaboratories, Connecticut, USA) was diluted to 1000 HAU/μl(hemagglutinin units per microliter) in RPMI media (RPMI+10%FCS+L-glutamine). The volume of diluted virus required was based on theproportion of pDCs in the purified PBMCs, so that there is at least 1HAU/pDC (i.e., each well should contain 1×10⁶ PBMCs, and if you have0.3% pDCs, each well will contain 3000 pDCs. In that case, 4-5 μL ofvirus at 1000 HAU/μL will be added to each well).

PBMCs prepared from the buffy coat were centrifuged at 900 rpm for 10min and resuspended at 5,000 cells/μL (this will provide for 1×10⁶ cellsper well in a volume of 200 μL) in RPMI+10% FCS+L-glutamine). The cellsplus flu virus were plated in 96-well U-bottom plates, and incubated at37° C.+5% CO₂ for 24 h. Following incubation for 24 h, the cells formeda pellet at the bottom of the wells and have formed clusters. Thesupernatant was harvested from the wells by careful pipetting, avoidingcell pellets at the bottom of the wells. Combined supernatants werecentrifuged in 50 mL conical tubes at 900 rpm for 10 min to pellet anyresidual cells and other culture debris. PBMC-flu supernatants,containing a complex mixture of IFNα, were pooled, mixed, and stored at−80° C. in 0.5 ml aliquots until use.

Cytopathic Effect (CPE) Inhibition Assay. CPE Materials: Dulbecco'sModified Eagle's Medium (DMEM) “complete”: DMEM with phenol red+10%FCS+2 mM L-glutamine+penicillin+streptomycin+β-2-mercaptoethanol (β-me),96-well flat-bottom tissue culture plates, A549 cells (ATCC CCL-185);these cells are cultured in Ham's F12K medium+10% FCS+2 mML-glutamine+penicillin+streptomycin+500-800 μg G418, 1×PBS, 1× trypsin,“Intron A” (IFNα-2b, Schering-Plough) controls, test samples (SLE serum,recombinant IFNα subtypes) with/without hybridoma supernatant orpurchased polyclonal/monoclonal antibody preparations, EMC virus stocks(prepared from the murine encephalomyocarditis virus (EMCV) adapted totissue culture on Vero cell monolayers; the ATCC product number for thisviral stock is VR-129B, and the product number for Vero cells isCCL-81).

Crystal violet stock solution: 1.25 mg NaCl+3.75 mg crystal violet+775mL formaldehyde/ethanol solution (which is prepared with 75 mLformaldehyde, 750 mL 95% ethanol, and 1500 mL distilled water) werestirred for 20 min, then filtered through a 0.45 micron filter andstored for not longer than 3 months. A working solution of Crystalviolet was prepared by diluting the stock solution 1:10 with theformaldehyde/ethanol solution. The crystal violet solutions were storedat room temperature.

CPE Methods. A schematic diagram of the Cytopathic Effect InhibitionAssay (CPE) is shown in FIG. 1. CPE assays were performed in triplicatewells in 96-well flat-bottom plates. For each assay type, it was usefulto incorporate positive control wells containing intron A(Schering-Plough) samples of varying concentrations and negative controlwells containing only cells+media. Adherent A549 cells were harvestedfrom flasks by removing the culture media, washing once with PBS, andtrypsinization. Trypsinization was stopped by adding DMEM “complete” tothe flask. The tyrpsinized cells were collected from the flask,centrifuged, resuspended and counted. Their concentration was adjustedto 600,000 cells/mL in complete DMEM. The volume of the wells for theassay was 150 μL (prior to virus addition) and 200 μL (following virusaddition). 50 μL of the volume was cells, of which 15,000 cells (50 μLof the 300,000/mL cell suspension) was added per well.

To assay for viral inhibition by recombinant IFNα. 100 μL of variousconcentrations of IFNα solution (in DMEM complete) was added intriplicate wells. 50 μL of cells was added and incubated for S hours at37° C.+5% CO2. After this time period, 50 μL of EMC virus diluted50-fold from stock was added and incubation continued for 48 h at 37°C.+5% CO₂.

To assay for viral inhibition by SLE serum. 50 μL (e.g., no dilution) or25 μL (e.g., 2× dilution) of SLE serum was placed in triplicate wells.The volume was adjusted to 100 μL by adding DMEM without FCS (note: anytime serum samples of any type are added to wells using this assay, DMEMwithout FCS was used) and then 50 μL of cells was added and incubatedfor 4 h at 37° C.+5% CO₂. After this time period, 50 μL of EMC virusdiluted 50-fold from stock (this concentration was determined, for ourpreparation, as the minimal amount of stock able to kill all cells in 48hours) was added and incubation continued for 48 h at 37° C.+5% CO₂.

To assay for viral inhibition by PBMC-flu supernatants. 50 μL (e.g., nodilution) or serial dilutions of PBMC-flu supernatant was added intriplicate wells. The volume of each well was adjusted to 100 μL byadding DMEM with FCS, and then 50 μL of cells was added and incubatedfor 4 h at 37° C.+5% CO₂. After this time period, 50 μL of EMC virusdiluted 50-fold from stock was added and incubation continued for 48 hat 37° C.+5% CO₂.

To assay for antibody-mediated blockade of viral inhibition byrecombinant IFNα, SLE serum, or PBMC-flu supernatants usingcommercially-available Abs, mouse serum, or fusion supernatants, 50 μLof either: (i) DMEM without FCS containing commercial polyclonal ormonoclonal Ab preparations; (ii) 50 μL of mouse serum; or (iii) 50 μL ofhybridoma supernatant was added to bring the total volume of each wellat this point to 100 μL. The plates were incubated for 1.5 to 2 h at 37°C.+5% CO₂. 50 μL of cells was added to each well and incubated for 5 hat 37° C.+5% CO₂. After this time period, 50 μL of EMC virus diluted50-fold from stock was added and incubation was continued for 48 h at37° C.+5% CO₂.

After 48 hour incubation period in the above CPE assays, all media wascarefully removed from the wells using a multichannel pipet. 50 μL ofcrystal violet was added and allowed to stain for 4-6 min. The crystalviolet was carefully removed and 200 μL of distilled water was added andimmediately removed. The plates were allowed to dry for at least 30 min,and then read on an ELISA plate reader at an OD of 570 nm.

To obtain the percentage of blockade (based upon the ability of theincluded control antibody or antibody contained in the hybridomasupernatants to inhibit IFNα-mediated protection against cell death),the data was normalized in the context of the “negative control” (IFNαrecombinant, SLE serum, or PBMC-flu supernatant+cells+virus) being 100%viability (0% cell death) and the “positive control” (cells+virus only)being 0% viability (100% cell death) using the Prism 4.0 for Macintosh,Version 4.0A software (GraphPad Software, Inc., San Diego, Calif.) andthe Normalize algorithm to adjust all of the values to percentagesaccording to the controls.

Reporter Gene (RG) Assay. RG Materials. Dulbecco's Modified Eagle'sMedium (DMEM), no phenol red or supplements; Dulbecco's Modified Eagle'sMedium (DMEM) “complete”: DMEM with phenol red+10% FCS+2 mML-glutamine+penicillin+streptomycin+2-me (β-me); Dulbecco's ModifiedEagles Medium (DMEM) prepared to contain everything listed above exceptFCS; ViewPlate-96, white, tissue culture-treated (PerkinElmer LifeSciences); 93D7 cells (A549 transfected to express luciferase driven bythe type I IFN-inducible MxA promoter); these cells are cultured inHam's F12K medium+10% FCS+2 mML-glutamine+penicillin+streptomycin+500-800 μg G418; 1×PBS; 1× trypsin;“intron A” (IFNα 2b, Schering-Plough) controls; test samples (SLE serum,recombinant IFNα subtypes) with/without hybridoma supernatant orpurchased polyclonal/monoclonal antibody preparations; Britelite™luminescence reporter gene assay kit (PerkinElmer Life Sciences).

RG Methods. A luciferase-based reporter gene assay was utilized toevaluate the ability of the anti-IFNα MAbs to neutralize the bioactivityof recombinant IFNα subtypes, leukocyte IFN and PBMC-flu. A schematicdiagram of the RG assay is shown in FIG. 1. The 93D7 cell line, whichwas derived by stable transfection of the A549 cell line (CLL-185, ATCC)with an IFN-inducible construct (MxA promoter/luciferase fusion) waskindly provided by Dr. Guenther Adolf (Boehringer-Ingelheim GmbH,Austria). The MxA promoter/luc fusion vector includes a 1.6 Kb BamHIfragment containing the murine MxA promoter and IFN response elementsexcised from pSP64-Mxp(PstI-PvuII)-rβglo (Lleonart et al. (1990)Biotechnology 8:1263-1267) and inserted upstream of a luciferase codingsequence.

RG assays were performed in triplicate wells in opaque 96-well,flat-bottom (ViewPlates™, white walls, clear bottom; PerkinElmer). Forevery assay type, it is preferable to incorporate positive control wellscontaining intron A (Schering-Plough) samples of varying concentrationsand negative control wells containing only cells+media.

Specifically, adherent 93D7 cells were harvested from flasks by removingthe culture media, washing once with PBS, and trypsinization.Trypsinization was stopped by adding DMEM “complete” to the flask. Thecells were collected from the flask, centrifuged, resuspended andcounted. Their concentration was adjusted to 600,000/mL in completeDMEM.

Serum from immunized mice, hybridoma supernatants or purified anti-IFNαMAbs was pre-incubated with recombinant IFN subtypes, leukocyte IFN orPBMC-flu in 100 μl/well volumes for 1.5 hours at 37° C.+5% CO₂, afterwhich 93D7 cells (50 μL of the 600,000/mL cell suspension=30,000 cells)was added per well and incubation was continued for an additional 5hours. The final volume of the wells for the assay was 150 μL. Assayswere then developed using the Britelite™ luminescence reporter genesystem (Perkin Elmer) and read on a Wallac Microbeta Trilux™scintillation and luminescence counter within 15 minutes of adding thesubstrate.

To assay for MxA induction by recombinant IFNα. The concentration of asolution (in DMEM complete) of IFNα was adjusted to contain the amountto be placed in each well in 100 μL. 100 μL of IFNα was placed intriplicate wells and then 50 μL of cells were added and incubated for 5hours at 37° C.+5% CO₂.

To assay for MxA induction by SLE serum. 50 μL (e.g., no dilution) or 25μL (e.g., 2× dilution) of SLE serum was placed in triplicate wells. Thevolume/well was then adjusted to 100 μL by adding DMEM without FCS(note: any time serum samples of any type are added to wells using thisassay, DMEM without FCS will be used) and then 50 μL of cells was addedand incubated for 5 hours at 37° C.+5% CO₂.

To assay for blockade of MxA induction by PBMC-flu supernatants. 50 μL(e.g., no dilution) or serial dilutions of PBMC-flu supernatant wasplaced in triplicate wells. The volume of each well was then adjusted to100 μL by adding DMEM with FCS, followed by the addition of 50 μL ofcells and incubation for 5 hours at 37° C.+5% CO₂.

To assay for blockade of MxA induction by recombinant IFNα, SLE serum,or PBMC-flu supernatants using commercially-available Abs, mouse serum,or fusion supernatants. 50 μL or a desired dilution of recombinant IFNα,SLE serum, or PBMC-flu was added per well. 50 μL of either: (i) DMEMwithout FCS containing commercial polyclonal or monoclonal antibodypreparations; (ii) 50 μL of mouse serum; or (iii) 50 μL of hybridomasupernatant was then added to each well to bring the total volume ofeach well to 100 μL. The plates were incubated for 1.5 hours at 37°C.+5% CO₂. After this time, 50 μL of cells was added and incubationcontinued for 5 hours at 37° C.+5% CO₂.

Britelite™ kit reagents/developing reagents (substrate vials, substratebuffer, uncolored DMEM) were set at room temperature 40 min prior toassay development. At 30 min pre-development, the assay plates wereplaced at room temperature. At 10 min pre-development, the lyophilizedsubstrate was reconstituted with buffer (10 mL per vial).

After the 5 hour incubation period, all media was carefully removed fromthe wells using a multichannel pipet. Next, an adhesive white blockerwas fixed to the bottom of the ViewPlate™. 90 μL of DMEM (without phenolred) was added per well. 90 μL of reconstituted Britelite™ reagent wasadded to each well, making sure to pipet up and down twice (but withouteither splashing the well contents onto the sides of the wells orcreating air bubbles) for thorough mixing of the reagent and the media.This was performed as quickly yet precisely as possible. The plate wassealed with a clear adhesive sealing strip. Within a span of greaterthan 1 min but not more than 15 min, the luminescence intensity of theplate(s) was read using the Wallac Microbeta Trilux™.

To obtain the percentage of blockade (based upon the ability of theincluded control antibody or antibody contained in the hybridomasupernatants to negate MxA-luciferase induction), the data wasnormalized in the context of the “positive control” (IFNα recombinant,SLE serum, or PBMC-flu supernatant+ cells) being 100% IFNα activity andthe “negative control” (cells in media only) being 0% IFNα activity,using the software Prism (GraphPad Software, Inc., San Diego, Calif.)and the Normalize algorithm to adjust all of the values to percentagesaccording to the controls.

Example 1 Immunization and Selection of Monoclonal Antibody Cell Lines

A flow chart of the IFNα MAb development scheme is shown in FIG. 2.Groups of five 6-8 week old Balb/c female mice (Harlan) were immunizedwith 5-10 μg each natural leukocyte IFNα (1-2396, Lot #111K1603, Sigma)and/or a cocktail of recombinant proteins (5-10 μg each of the threerecombinant IFN a subtypes A, B2, and F (obtained from PBL BiomedicalLaboratories “PBL”)) in MPL®+TDM emulsion (Sigma #M6536) at two to threeweek intervals according to the schedules indicated in Table 1, below.The MPL®+TDM emulsion is a Ribi Adjuvant system consisting ofmonophosphoryl-lipid A (MPL: detoxified endotoxin from S. Minnesota) andtrehalose dicorynomycolate (TDM) in a 2% oil (squalene)-Tween 80-wateremulsion. Antigen was administered via intraperitoneal (i.p.) orsubcutaneous (s.c.) routes. Pre-fusion screening of serum collected fromthe mice was done at three titers (1:200, 1:2000, and 1:20,000) usingthe reporter gene (RG) assay, based upon a MxA-luciferase fusion proteinvia activation of the Type I IFN receptor, to detect blockade of IFNαbioactivity. Serum was collected from the mice via retro-orbital bleedseven days following the third boost and screen for neutralization ofPBMC-flu bioactivity using the reporter gene (RG) assay described above.Mice exhibiting titers of at least 1:2000 for >50% neutralization wererested for four weeks and then given a final boost (either i.v. or i.p.)of 2.5 μg leukocyte IFN prior to splenocyte fusion with the murinemyeloma Sp2/0-Ag14 (CRL-8287, ATCC) three days later. Fusions wereperformed in 50% PEG 1500 (Roche), and 1×HAT supplement (Sigma) inDMEM+15% FCS was used for hybridoma selection. Culture supernatants werescreened 10-14 days later for neutralization of PBMC-flu. Based in theMxA/luc reporter gene (RG) bioassay, described above, of the miceimmunized, 17 were able to neutralize the PBMC-flu supernatant by atleast 50% at titers of 1:200, among these, 14 could neutralize at ≧50%at 1:2000 dilutions, and 3 continued to neutralize at titers up to1:20000.

TABLE 1 Immunization and Selection of Monoclonal Antibody Cell LinesProtocol 1 Protocol 2 Protocol 3 Protocol 4 Protocol 5 Balb/c miceBalb/c mice Balb/c mice B6 mice B6 mice Initiation At day 0 At day 0 Atday 0 At day 0 At day 0 10 μg natural 10 μg IFNα 5 μg natural 2.5 μgIFNα F 4 μg IFNα IFNα A, B2 and F IFNα, split 5 mice i.p. B2 5 mice i.p.5 mice i.p. i.p. 2.5 μg IFNα F 5 mice i.p. 5 mice s.c. 5 mice s.c. ands.c. and B2 3 groups of 4 5 mice i.p. mice (received adeno- mIFNα at Day−2, +2 or 1^(st) boost) 1^(st) boost At 2 weeks At 2 weeks At 2 weeks At2 weeks At 1.5 weeks 5 μg natural 5 μg IFNα A, 2.5 μg 2 μg IFNα F 2 μgIFNα A IFNα B2 and F natural IFNα, 5 mice i.p. 5 mice i.p. 5 mice i.p. 5mice i.p. split i.p. 2 μg IFNα F 5 mice s.c. 5 mice s.c. and s.c. and B25 mice i.p. 2^(nd) boost At 5 weeks At 5 weeks At 5 weeks At 5 weeks At4 weeks 5 μg natural 5 μg IFNα A, 1.5 μg 4 μg IFNα F 2 μg IFNα F IFNα B2and F natural IFNα, 5 mice i.p. 5 mice i.p. 5 mice i.p. 5 mice i.p.split i.p. 4 μg IFNα F 5 mice s.c. 5 mice s.c. and s.c. and B2 5 micei.p. 3^(rd) boost At 10.5 At 9 weeks At 8.5 weeks At 7 weeks N/A weeks 2μg IFNα A, 2 μg natural 2 μg IFNα F 2.5 μg B2 and F IFNα, i.p. 5 micei.p. natural IFNα All mice i.p. 2 μg IFNα F All mice i.p. and B2 5 micei.p. 4^(th) boost At 16.5 At 13.5 N/A At 9 weeks N/A weeks weeks 2.5 μgIFNα A 2.5 μg 2.5 μg All mice i.p. natural IFNα natural IFNα All micei.p. All mice i.p. Note: Mice from protocols 1, 2 and 3 were used tomake fusions 1 through 6. The fusions were perfomed pooling the cellsfrom 2-3 mice within a protocol. Two mice (initially immunized with IFNαF in Protocol 6 were pooled to make fusion 8). In Protocol 5, two micewere pooled to make fusion 7, and three were pooled to make fusion 9.

Production and purification of monoclonal antibodies. As describedabove, mice were identified as candidates for fusion based upon theability of their serum to neutralize the complex mixture of IFNαsubtypes present in PBMC-flu. A series of 8 fusions were performed withsplenocytes harvested from mice with acceptable serum titers.Splenocytes were fused with the Sp2/0-Ag14 murine myeloma cell line(ATCC Number CRL-1581, which was selected since it is unable to expressendogenously-derived Ig chains), plated in 96-well flat-bottom tissueculture plates, and incubated for 12-15 days prior to screening ofsupernatants in order to detect a polyclonal antibody response via theRG assay protocol described above. Specifically, serum samples from theimmunized mice were preincubated with supernatant from flu-infected PBMCfor 1 hour at 37° C., after which the 93D7 cells were added for anadditional 5 hours. At 5 hours, assays were developed and read on aluminescence counter. A summary of the first 8 fusions is shown in Table2, below. Supernatants from the 8 fusions were screen from 3911 primarywells, and eight candidates (ACO-1 through 8), each isolated from Fusion4, were identified based on their capacities to consistently demonstrateany visible decrease in PBMC flu (diluted 640-fold) mediated activationof MxA/luc production in the RG bioassay described herein. Hybridomacell lines were subcloned by limiting dilution. Hybridomas producinganti-IFN-α MAbs were adapted to growth in Gibco PFHM-II (Invitrogen) andcultured in Integra CELLine flasks (Becton Dickinson). Supernatants werecollected from the cell compartments every 5-7 days and frozen at −80°C. The MAbs were then purified from 50 ml batches of supernatant viaFPLC over protein A columns followed by dialysis into PBS. The purifiedMAbs were aliquotted and stored at −80° C. ACO-1, 2, 3, 4, 5, and 8 wereisotyped by ELISA as IgG2a (ACO-1), IgG2b (ACO-2), and IgG1 (ACO-3, 4,5, 6 and 8). All of these candidates derived from fusions of splenocytesfrom mice initially immunized with leukocyte IFN or a mixture of IFN-αA, B2, and F recombinant proteins followed by a pre-fusion boost withleukocyte IFN; fusions performed from mice administered only therecombinant IFN-α subtypes failed to yield any candidates able toneutralize PBMC-flu.

TABLE 2 Summary of Fusions Fusion 1 2 3 4 5 6 7 8 Totals Screened 4351644 560 396 136 217 191 332 3911 Selected 12 66 2 25 0 29 5 15 154Subcloned 3 13 0 8 0 2 0 0 26 Candidates 0 0 0 6 0 2 0 0 8

Example 2 Neutralization of Commercially-Available Leukocyte IFNα orPBMC-Flu Supernatant Bioactivity by ACO-1 ACO-2, ACO-3, ACO-4 and ACO-5

The anti-IFN-α MAbs shown to strongly bind and neutralize at least oneIFN-α subtype were selected to examine their abilities to neutralizenaturally-derived IFN preparations, which are known to contain a broadvariety of IFN-α subtypes. For these studies, ACO-1 through 5 weretitrated in the RG bioassay against both commercially-availableleukocyte IFN and PBMC-flu supernatant prepared as describe above.ACO-1, 2, and 3 blocked leukocyte IFN bioactivity by at least 50% at allthree MAb amounts tested (200, 20, and 2 ng) (FIG. 3 a); ACO-4 achievedslightly more than 50% neutralization only when 200 ng was tested. Incomparison, ACO-5 performed poorly against leukocyte IFN, maximallyblocking less than 10% of the assay signal.

Comparative inhibition of various dilutions of PBMC-flu supernatant bydefined concentrations of the monoclonal antibodies was performed usingthe RG assay described above. The absolute concentration of IFNα in theFlu/PBMC supernatant is unknown, so only relative neutralizing capacityis assessed in this study. When the five MAbs were titrated (2000, 200,20, and 2 ng) against PBMC-flu however, ACO-5 was able to neutralizebioactivity by at least 50% at all but the lowest antibody amountstested (FIG. 3 b). ACO-1 exhibited the greatest potency when tested onPBMC-flu, blocking by at least 50% at all four titrated MAb amounts. Thevariance in neutralization of leukocyte IFN and PBMC-flu by ACO-5 islikely attributable to different IFN-α subtypes and/or their relativeconcentrations present in the two separate IFN sources used in ourassays.

Example 3 Inhibition of Recombinant IFNα Subtype Bioactivity by ACO-1,ACO-2, ACO-3 ACO-4, ACO-5 ACO-6 and ACO-8

The IFNα-neutralizing candidates ACO-1 through 6 and ACO-8 were screenedby both the RG assay as well as the traditional cytopathic effect (CPE)inhibition assay for neutralization of 15 recombinant IFNα subtypes aswell as for neutralization of IFNβ. Recombinant IFN-α subtype proteinswere obtained from PBL Biomedical Laboratories (Piscataway, N.J.);info@interferonsource.com, hereinafter “PBL”). The specific activities,as determined by the manufacturer, are shown in Table 3.

TABLE 3 Recombinant human IFN-α subtypes employed in the antibodycharacterizations. IFNα RG RG CPE CPE Protein Specific plateau middleplateau middle subtype activity Product Lot # RG_(max) (pg of (pg of (pgof (pg of (gene) (U/mg) # (PBL) (PBL) Units IFN) IFN) IFN) IFN) A (2a) 3.8 × 10⁸ 11100-1 2167 47.5 125 62.5 50 25 2 (2b) 2.77 × 10⁸ 11105-12122 8.31 30 15 25 12.5 B2 (8) 4.63 × 10⁸ 11115-1 2168 3.7 8 5 5 2.5 C(10) 2.31 × 10⁸ 11120-1 2118 3.47 15 4 5 2.5 D  7.5 × 10⁷ 11125-1 2403150 2000 750 750 375 [Val¹¹⁴] (1) F (21)  3.6 × 10⁸ 11130-9 2169 72 20062.5 25 12.5 G (5) 2.33 × 10⁸ 11135-1 2104 116.5 500 62.5 50 25 H2 (14)1.05 × 10⁸ 11145-1 2528 131.25 1250 200 25 12.5 I (17)  1.4 × 10⁸11150-1 1770 8.75 62.5 15 10 5 J1 (7)  2.6 × 10⁸ 11160-1 2105 260 1000200 20 10 K (6) 1.48 × 10⁸ 11165-1 1359 92.5 62.5 155 25 12.5 1  1.4 ×10⁸ 11175-1 2106 17.5 125 15 200 100 [Ala¹¹⁴] (D) 4a (4a) 2.12 × 10⁸11177-1 2180 10 10 2 25 12.5 4b (4b)  1.8 × 10⁸ 11180-1 1424 45 250 31100 50 WA  2.4 × 10⁸ 11190-1 2107 30 125 15 25 12.5 IFN-β 8.23 × 10⁷2558 6.56

The neutralizing units (U) provided by the manufacturer has beenassigned via an assay measuring the ability of the given subtypes toneutralize 50% (identified as 1 U/ml) of the cytopathic effect producedby vesicular stomatitis virus on bovine MDBK cells. Given that IFN-αpotencies in bioassays are influenced by numerous variables (includingassay type, individually prepared batches, and minor techniquevariations from one laboratory to another), as well as the fact thatinternationally-recognized standards for each subtype are unavailable,single lot numbers were consistently employed in these studies. Themanufacturer-defined U of each recombinant that would yield maximalresponse in the RG bioassay (RG_(max)) was determined. Purified ACO-1,2, 3, 4, 5, 6 and 8 were then titrated in the presence of these IFN-αsubtype quantities in the RG bioassay. IFN-β (specific activity=8.23×10⁷U/mg) was obtained from PBL.

Representative RG bioassay data for the titration of ACO-1 against theRG_(max) values of the fifteen IFN-α subtypes is shown in FIG. 4 a. Asdescribed in the legend, numerical values were assigned to each ACO-1titration against the designated subtypes based upon the EC₅₀ valuesdetermined for each. ACO-1 was unable to neutralize either IFN-α D orIFN-α1, whereas the other thirteen subtypes could be neutralized atvarious antibody concentrations. The EC₅₀ results in ng/ml for allACO-1, 2, 3, 4, 5 and 8 IFN-α-neutralizing MAbs are provided in Table 4.The percentage neutralization is shown in Table 5. Accordingly, ACO-1and 2 appear similar in their capacities to neutralize twelve subtypes(IFN-α A, 2, B2, C, F, G, H2, I, J1, 4a, 4b, and WA) at concentrationsof less than 300 ng/ml of antibody; ACO-1 also neutralized IFN-α K tothis extent, though ACO-2 did not. ACO-3 and 4 neutralized nine (IFN-αA, 2, B2, C, I, J1, K, 4a, and WA) and six (IFN-α A, 2, B2, C, I, J1,and 4a) subtypes with less than 300 ng/ml, respectively. ACO-8neutralized four subtypes (IFN-α2, 1, 4a, and 4b) given the antibodyconcentration constraint, while ACO-5 strongly neutralized only three(IFN-α A, 2, and WA). None of the MAbs were able to neutralize IFN-β(FIG. 4 b).

TABLE 4 Neutralization of recombinant IFN-α subtypes by ACO-1, 2, 3, 4,5 and 8 (EC₅₀ ng/ml). IFN-α subtype ACO-1 ACO-2 ACO-3 ACO-4 ACO-5 ACO-8A 23.15 64.17 4.82 23.57 3.61 335.5 2 16.52 38.57 0.31 19.21 0.23 180.4B2 8.09 6.09 1.01 172.3 — 308.2 C 0.56 2.19 3.22 152 539.3 — D — — — — —373 F 145.8 54.05 — — 704.7 349.8 G 27.07 43.29 — — 465.8 — H2 153.3123.7 637.1 — 604.7 — I 13.5 8.4 6.32 147.1 528.1 342.6 J1 168.3 212.6206.6 — 646.5 488 K 235.2 352.9 239.5 475.3 444.8 — 1 — — — — 576.7211.2 4a 2.11 5.6 0.04 55.59 597.6 134.4 4b 5.43 7.27 — — 669.9 271.8 WA84.85 106.8 250.4 — 140.5 —

TABLE 5 Percentage neutralization of RG_(max) IFN amounts by 2micrograms/mL of antibody IFN-α subtype ACO-1 ACO-2 ACO-3 ACO-4 ACO-5ACO-8 A 93.6 89.4 86.7 59.5 92.7 73.1 2 90.3 88 88.8 85.3 95.8 77.6 B287 87.5 87 75.2 29.2 77 C 91.4 92.9 85.8 70.9 28.4 30.5 D 12.5 20.8 08.2 30.4 42 F 83.7 88 0 1 24.9 55.2 G 92.7 95 0 0 59.6 37.3 H2 77.5 8722.6 6.5 16.5 25.4 I 87.1 93.3 79.6 46.7 58.9 68.5 J1 83.9 81.9 45.2 2.920.5 31.8 K 77.9 71.2 60 12.9 53 25.2 1 0 13.5 1 5.1 46.7 75.7 4a 67.867.4 64 58.3 31.6 71.3 4b 93.7 92.4 9 4.8 24.2 63.2 WA 86.9 84 55.5 5.485.2 14.3 IFN-β 9 0 6.5 1 0 0

The CPE assay was set up similarly to the RG assay with untransfectedA549 cells (ATCC Number CCL-185, a human lung carcinoma cell line):assays are performed in standard 96-well flat-bottom tissue cultureplates. Following pre-incubation (1 hour at 37° C.) of antibodies withthe IFNα subtypes and addition of cells 5 hours later, mouseencephalomyocarditis virus (EMCV) was added and the cells were incubatedfor 48 hours prior to staining with crystal violet for assessment oflive cells remaining. For both the RG and CPE assays, the quantities ofeach IFNα subtype used were determined via prior titrations of therecombinant IFNα proteins to yield either maximal MxA-luciferaseinduction (RG) or protection from cell death (CPE) in the assay. Thedata shown in Table 6 represents the percentage of bioactivity blockadedemonstrated by each ACO monoclonal antibody against the respective IFNαsubtypes (corresponding genes encoding the subtypes are indicated inparentheses) in the CPE assay. For both the CPE assay, the quantities ofeach IFNα subtype used were determined via prior titrations of therecombinant IFNα proteins to yield either maximal protection from celldeath (CPE). N/D=not determined. As shown, ACO-1 and ACO-2 are capableof blocking the largest number of IFNα subtypes at levels of ≧90% underthe assay conditions, whereas ACO-6 is the most restricted. In mostcases, the outcomes of the RG and CPE assays correlate with one another.

TABLE 6 Percentage Neutralization of IFNα subtype activity by Mabs inthe CPE Assay. IFN-α ACO-1 ACO-2 ACO-3 ACO-4 ACO-5 ACO-6 A (2a) 95.781.4 93.9 94.6 90.0 0.0 2 (2b) 80.1 75.8 96.6 92.4 93.9 0.0 B2 (8) 98.898.6 96.4 49.6 13.0 0.0 C (10) 99.4 98.2 92.1 65.1 21.9 0.0 D (1) 33.038.5 0.0 11.3 0.0 0.0 F (21) 96.6 95.4 0.0 23.5 34.5 0.0 G (5) 99.0 97.40.0 0.0 80.8 0.0 H2 (14) 95.9 98.3 92.6 90.7 78.9 0.0 I (17) 98.0 99.895.0 85.1 56.2 0.0 J1 (7) 98.1 97.3 92.8 47.1 61.1 0.0 K (6) 98.3 99.590.1 82.1 85.3 0.0 4a (4a) 82.9 83.6 81.3 N/D 0.0 0.0 4b (4b) 98.7 96.70.0 0.0 53.7 0.0 WA (16) 88.7 94.6 91.0 N/D 100 0.0 1 (1) 16.7 22.9 15.911.9 64.3 0.0 IFN-α N/D N/D N/D N/D N/D N/D

Example 4 Multiplex Analysis of ACO-1 ACO-2, ACO-3 ACO-4, ACO-5, andACO-6

Multiplex analysis was conducted to assess whether spatially distinctbinding domains were involved. The ACO antibodies were analyzedcombinatorially for their abilities to simultaneously bind IFNα-A viamultiplex analysis on a Luminex™ 100 system. Beads coupled withunlabeled ACO antibodies (Capture) were incubated with recombinantIFNα-A at the concentration indicated and then exposed to PE-labeled ACOantibodies (Reporter). This examination revealed that ACO-5 canmultiplex with any of ACOs-1, -2, -3, and -4 (see light shading in FIG.5). Multiplexing additionally occurs when ACO-4 is employed as a captureantibody and ACO-3 as a reporter antibody. Accordingly, ACO-5 binds aspatially distinct domain of IFNα-A than that bound by ACO-1, 2, -3 and-4. Similarly, ACO-3 and ACO-4 bind spatially distinct domains ofIFNα-A. Results with ACO-6 were negative in all cases.

Example 5 Affinity Determinations for ACO-1. ACO-2, ACO-3, ACO-4 ACO-5,and ACO-6

Kinetic analysis of the ACO antibodies against IFNα-A was performedusing Biacore 2000 and 3000 optical biosensors equipped with CM5 sensorchips and equilibrated with 10 mM HBEPES, 150 mM NaCl, 0.005% P20, 0.1mg/ml BSA, pH 7.4 at 25° C. For each of ACOs 1 through 6, antibodieswere first buffer-exchanged from Tris-glycine buffer to 10 mM sodiumacetate buffer, pH 5.0 using a fast desalting column and thenimmobilized on three flow cell surfaces using standard amine-couplingchemistry, while the fourth was left unmodified to serve as a reference.Final MAb immobilization densities ranged from 500-1100 RU (responseunits). Binding responses were monitored as IFNα-A was flowed intitrated amounts (0, 0.31. 0.93. 2.78. 8.33. 25.0 and 75.0 nM) over theantibody and reference flow cells at a rate of 50 μl/min. Association ofthe Ab/Ag complex was monitored for four minutes and the dissociationwas monitored for twelve minutes. The surfaces were regenerated with1/1000 H₃PO₄ (except ACO-5, which required 1/200 H₃PO₄) at the end ofeach binding cycle. Assays were performed in triplicate. The results areshown in Table 7. The K_(D) values for the five anti-IFN-α MAbs covereda range inversely proportional to the breadth of IFN-α subtypesneutralized by each MAb as well as their potency in blocking leukocyteIFN and PBMC-flu bioactivity. ACO-1 exhibited the lowest affinity(5.61×10⁻⁹ M), while ACO-5 exhibited a 14-fold higher affinity(4.00×10⁻¹⁰ M). ACO-6 does not bind to IFNα-A and therefore no rateswere obtainable.

TABLE 7 Biacore Kinetic Analysis of ACO-1 through ACO-6 with IFN-α AAntibody K_(a) (M⁻¹ s⁻¹) K_(d) (s⁻¹) K_(D) (M) ACO-1 7.86 × 10⁴ 4.41 ×10⁻⁴ 5.61 × 10⁻⁹  ACO-2 9.87 × 10⁴ 1.90 × 10⁻⁴ 1.92 × 10⁻⁹  ACO-3 5.12 ×10⁵ 2.36 × 10⁻⁴ 4.61 × 10⁻¹⁰ ACO-4 5.82 × 10⁵ 2.74 × 10⁻⁴ 4.71 × 10⁻¹⁰ACO-5 5.25 × 10⁵ 2.10 × 10⁻⁴ 4.00 × 10⁻¹⁰ ACO-6 N/A N/A N/A

Example 6 Solid-Phase Binding of IFNα Subtypes by ACO-1, ACO-2, ACO-3,ACO-4, ACO-5, and ACO-6

Solid-phase binding of all 15 IFNα subtypes was evaluated for screeningof MAb specificity by ELISA assay. Briefly, 50 μl/well of 1 μg/ml of arecombinant IFNα protein subtype were coated overnight at 4° C. on ELISAplates (NUNC MaxiSorp™) plates. Coated plates were blocked with PBS+1%BSA and incubated with 50 μl of 25 ng ACO candidate MAb in PBS for 1hour at 37° C. The assays were developed by incubation with 50 μl/wellof an HRP conjugated goat anti-mouse-IgG (Jackson ImmunoResearch) atroom temperature for 30 minutes followed by incubation with 100 μl/wellof TMB substrate solution (Zymed) for 15 minutes. The reaction wasstopped with 100 μl/well of 1 N HCl and read at OD₄₅₀ on an ELISA platereader. Binding percentages were calculated by normalizing backgroundsignal values with maximal signal values across the assay (observed forIFNα-4-a). The results are shown in Table 8 and FIG. 6. Both ACO-1 and 2bound the identical twelve IFN-α subtypes that they effectivelyneutralized in the RG bioassay at signals at least 2-fold greater thanisotype-matched controls; binding of IFN-α subtypes B2, K, 4a, and 4bdemonstrated signals more than 20-fold over controls. Differencesbetween binding and neutralization capacities were observed, however,among ACO-3, 4, 5, and 8. In the case of ACO-3, the ELISA signals forsubtypes B2, K, and 4a were the highest, despite the fact that the EC₅₀value for neutralization of IFN-α K was greater than 200-fold higherthat the EC₅₀ for IFN-α B2 and 4-fold higher for IFN-α4a; significantbinding of subtype J1, which was neutralized in the bioassay was notdetectable. The binding and neutralization profiles for ACO-4 and 5presented inverse relationships with one another. While ACO-4 and 5strongly bound one subtype each (IFN-α4a and 2, respectively), ACO-4 wasable to bind more subtypes than it neutralized, and ACO-5 was able toneutralize (albeit at high EC₅₀ values) more subtypes than it bound. Apotential explanation could lie in accessibility differences of thespecific epitopes recognized by these two MAbs in aqueous (RG) versussolid-phase (ELISA) assays. ACO-8 failed to strongly bind any of theIFN-α subtypes tested; it exhibited binding of less that 20-fold toIFN-α D, 1, and 4a. ACO-6 failed to bind to any of the subtypes.

TABLE 8 Binding of recombinant IFN-α subtypes by ACO-1, 2, 3, 4, 5, and8. IFN-α subtype ACO-1 ACO-2 ACO-3 ACO-4 ACO-5 ACO-8 A 6.4 9.8 8.4 7.21.7 1.0 2 8.7 7.0 9.8 9.0 3.4 1.1 B2 24.3  32.7  39.2  13.2  1.3 1.1 C5.2 5.4 3.2 2.2 1.5 1.0 D 1.2 1.4 1.0 2.5 1.7 3.7 F 5.3 10.8  1.0 2.81.4 1.4 G 7.4 8.5 0.9 2.4 1.3 1.1 H2 7.5 10.9  9.0 4.5 1.1 1.3 I 7.812.5  8.9 3.9 1.3 1.1 J1 2.1 3.0 1.8 2.6 1.4 1.1 K 23.2  22.3  27.7 15.1  1.4 1.1 1 1.3 1.1 1.0 2.7 1.6 2.8 4a 54.9  51.6  59.0  38.3  1.43.3 4b 22.2  26.1  1.0 2.6 1.4 1.4 WA 3.2 3.3 6.2 3.2 1.6 1.1Underlined indicates signals greater than 2-fold over matched controls.Bold indicates signals greater than 20-fold over matched controls.Signals of less that 2-fold over matched controls indicate insignificantbinding.

Example 7 Blockade of SLE Patient Serum Bioactivity by ACO-1, 2 and 3Monoclonal Antibodies

An antiviral assay was used to evaluate the ability of the anti-IFN-αMAbs to neutralize the protective activity of serum from SLE patientswith active disease against the death of A540 cells (CCL-185, ATCC) uponinfection with encephalomyocarditis virus (RMCV). The antibodiesexhibiting the broadest IFN-α subtype, leukocyte IFN, and PBMC-fluneutralization profiles (ACO-1, 2 and 3) were tested. The SLE serum wasobtained from four active SLE patients (identified as SLE-43, 133, 140and BC) selected upon the basis of IFN and granulopoiesis geneexpression signatures characterized from their blood mononuclear cells.SLE sera were screen for protection against viral infection in the CPEbioassay prior to MAb neutralization testing. The RG assay was notemployed in these analyses due to inhibition by serum factors of cellbinding to the ViewPlates™ during the comparatively shorter incubationperiod (5 hours) of the RG bioassay versus the CPE assay (48 hours).Vero cells (CCL-81, ATCC) were infected with EMCV (VR-129B, ATCC) toprepare working viral stocks from supernatants. Assays were performed intriplicate in tissue culture-treated, flat-bottom 96-well platedincubated at 37° C.+CO₂ with A549 cells (15,000 cells/well in 50 μleach) overnight. Anti-IFN-α MAbs and serum from SLE patients were thenadded to the plated (100 μl/well) and preincubated for 4 hours prior toaddition of EMCV diluted to the minimal concentration in 50 μl able tokill 100% of unprotected cells in 48 hours. Incubation was continued for48 hours, followed by staining with crystal violet and reading at OD₅₇₀in an ELISA plate reader. Controls were serum alone, media only (−), anda pan-neutralizing polyclonal antibody (pAb, rabbit anti-human IFN-α,PBL). The results shown in FIG. 7 a-d represent the mean of triplicates.Despite their lower affinities, ACO-1 and 2 were capable of neutralizingall four sera to some degree. ACO-3 was unable to block SLE-43, 140 orBC, The ability for relevant isotype control antibodies to block serumin some instances (IgG2b for SLE-43, all three isotypes for SLE-BC)likely resulted from natural variations in other serum constituents fromone patient to another that were cytotoxic to the cells employed in theassay.

Example 8 Cross Reactivity of ACO-1 and ACO-2 with primate IFN-α

To conduct preclinical safety/toxicology studies as a prelude to humanclinical trials, it is useful to identify an animal model whereendogenous IFN-α is reactive with the humanized anti-IFN-α monoclonalantibody. The ability of two candidates, murine anti-human IFN-α AbsACO-1 and ACO-2, to neutralize primate IFN-α were tested. Specifically,the ability of the antibodies to block induction of an MxA-luciferasereporter gene in A549 cells when stimulated with purified macaqueIFN-α4b (156 pg/well) was determined. As shown in FIG. 8, the antibodiesACO-1 and ACO-2 potently block reporter gene induction (A and B,respectively) while ACO-3 is unable to block even at high concentrations(C). Homology between human and Macaque IFN-α is highly conserved.Moreover, commercially available anti-human IFN-α antibodies have beenshown to cross-react with Rhesus and cynomologous homologs. These datasuggest that primates provide a suitable safety screening model.

Example 9 Sequence of ACO-1 Heavy and Light Chains

RT/PCR was performed using degenerate primer pools to amplify mRNA fromthe hybridoma expressing ACO-1. Heavy chain variable region mRNA wasamplified using a set of six degenerate primer pools (HA to HG) andlight chain variable region mRNA was amplified using a set of eightdegenerate primer pools (LA to LI). Amplification products were obtainedwith primer pools: HA, HB, HE, HF, LB, LC and LG. No PCR product wasamplified with pool LI, therefore the light chain is from the kappacluster. Each product was cloned and several clones from each sequenced.

Two different heavy chain sequences were identified. Pools HA and HFamplified a single sequence that codes for a truncated heavy chain witha stop codon at the end of Framework Region 3. It is therefore unlikelythat this heavy chain could form an antibody capable of binding toantigen.

Pools HB and HE amplified a single sequence that was different from thatof HA and HF and codes for a full length mouse V_(h) region as shown inFIG. 9. The full length heavy chain DNA sequence is SEQ ID NO:1, and thefull length amino acid sequence is SEQ ID NO:2. The DNA sequencesencoding CDRs V_(H)1 (TACACCTTCACCAACTACTGGATGCAC; SEQ ID NO:3), V_(H)2(GAGATTAATCCTAGCCACGGTCGTACTATCTACAATGAAAACTTCAAGAGC; SEQ ID NO:5) andV_(H)3 (GGGGGACTGGGACCCGCCTGGTTTGCTTAC; SEQ ID NO:7) are shown initalics while the amino acid sequences V_(H)1 (YTFTNYWMH; SEQ ID NO:4),V_(H)2 (EINPSHGRTIYNENFKS; SEQ ID NO:6) and V_(H)3 (GGLGPAWFAY; SEQ IDNO:8) are underlined.

Two light chain sequences were identified. Pools LB and LC amplified asingle sequence that aligned with the well documented, aberrant,truncated kappa light chain that is found in some hybridomas. Pool LGamplified a single sequence that was full-length and differed from thatamplified in pools LB and LC. The light chain sequence is shown in FIG.10. The full length light chain DNA sequence is SEQ ID NO:9, and thefull length amino acid sequence is SEQ ID NO:10. The DNA sequencesencoding CDRs V_(L)1 (AGTGCCGGCTCAAGTGTAGATTCCAGCTATTTGTAC; SEQ IDNO:11), V_(L)2 (AGC ACATCCAACCTGGCTTCT; SEQ ID NO:13) and V_(L)3(CATCAGTGGAGTAGTTACCCATTCACG; SEQ ID NO:15) are shown in italics, whilethe amino acid sequences V_(L)1 (SAGSSVDSSYLY; SEQ ID NO:12), V_(L)2(STSNLAS; SEQ ID NO:14) and V_(L)3 (HQWSSYPFT; SEQ ID NO:16) areunderlined.

The analysis of the sequences obtained from hybridoma ACO-1 issummarized in Table 9. The variable regions show high homology to theirclosest human germline sequences (67% to 65%) and the frameworksequences have close homologues in the human germline database.

TABLE 9 Clone ACO-1 H Chain L Chain CDR^(a) 1 Length V_(H)1: 9aa V_(L)1:12aa CDR 2 Length V_(H)2: 17aa V_(L)2: 7aa CDR 3 Length V_(H)3: 10aaV_(L)3: 9aa Mouse Germline J558.33 Vk ae4 Closest Human VH1-46 (67%) L6(65%) Germline^(b) Closest Human VH1-46/18/8/3/2 (80%) L20/A11/L6 (69%)FW1^(b) Closest Human VH1-46/69/18/2 (78%) L4/18a/018/012/L19/L18FW2^(b) L12/L11/08/02/L9/L8/L5 (80%) Closest Human VH1-69 (65%)A26/A10/A14 (75%) FW3^(b) Closest Human J^(b) J4 (92%) J2 (92%) ^(a)CDRdefinitions and sequence numbering according to Kabat ^(b)Germline ID(s)indicated followed by % homology

Example 10 Humanization of a Monoclonal Antibody and itsCharacterization

Humanized antibody is made by grafting the murinecomplementarity-determining regions into a human antibody framework(CDR-grafting) using methods known in the art (See Jones, et al., (1986)Nature, 321:522-525; Reichmann et al., (1988) Nature, 332:323-329;Presta (1992) Curr. Op. Struct. Biol., 2:593-596; and Clark (2000)Immunol.

Today 21: 397-402). The humanized antibody may be capable of the samebinding and functional parameters as the murine monoclonal antibodiesdescribed above.

Example 11 Treatment of SLE Using Humanized Monoclonal Antibody

Microarray analysis will be used to monitor the IFNα signature accordingto methods known in the art and described in Bennett, et al. (2003)supra and Baechler, et al. (2003) supra. This new tool will serve tostratify (i.e. positive IFNα signature inclusion criteria), as well asmonitor patients. Use of this analysis also is useful to determiningwhich patients are suitably treated by the compositions and methods ofthis invention. In one aspect, administration of an antibody of thisinvention will extinguish this signature. In one aspect, one of skill inthe art can determine when the object of a method of this invention ismet, and an effective amount of antibody has been delivered, when isdefined as the amount required the IFNα signature is suppressed by ≧50%for an effective amount of time, e.g. about 4 weeks.

An effective amount will be infused, e.g., from about 1 mg/kg, thesecond 2.5 mg/kg, the third 5 mg/kg antibody, and a fourth, ifnecessary, will be at 10 mg/kg. The “calculated optimal dose” for eachpatient is defined as the amount that can be safely administered andgives at least 50% suppression of the IFNα signature for about fourweeks.

Patients will be monitored weekly for IFNα signature. The time toreappearance of the IFNα signature will determine the dosing interval.For example, if the 1 mg/kg dose gives ≧50% signature reduction for only2 weeks, that patient would receive a 2nd does of 2.5 mg/kg. If weeklymonitoring revealed a ≧50% suppression of the signature for only 3weeks, the patient would receive their 3rd does of 5 mg/kg. A maximumdose of 10 mg/kg will be tested with the goal of identifying a dose thatgives ≧50% IFNα signature suppression for at least 4 weeks.

Efficacy can be measured by any acceptable method. Acceptable methodsinclude, but are not limited to microarray analysis of PBMCs (efficacyis established upon extinction of the interferon signature), flowcytometry of PBMCs (efficacy is established by increased T/B lymphocytecounts), decreased plasmacytosis and decreased presence of immatureneutrophils or cytokine multiplex analysis in serum by use of luminexanalysis.

Biological Deposits. The ACO-1, ACO-3 and ACO-6 hybridoma cell lineswere deposited with the American Type Culture Collection, 10801University Blvd., Manassas, Va. 20110-2209, USA (ATCC), and wereaccorded the deposit numbers listed in Table 10 below. A deposit ofACO-2 (labeled ACO2.2.1R) was deposited with the ATCC on Aug. 9, 2006.

TABLE 10 Hybridoma cell line ATCC Deposit No. Deposit Date ACO-1PTA-6557 Feb. 02, 2005 ACO-2 (deposit ACO2.2.1R) PTA-7778 Aug. 09, 2006ACO-3 PTA-6559 Feb. 08, 2005 ACO-6 PTA-6562 Feb. 08, 2005

These deposits were made under the provisions of the Budapest Treaty onthe International Recognition of the Deposit of Microorganisms for thePurpose of Patent Procedure and the Regulations there under (BudapestTreaty). This assures maintenance of a viable culture of the deposit for30 years from the date of deposit. The deposit will be made available byATCC under the terms of the Budapest Treaty, and subject to an agreementbetween Baylor Research Institute and ATCC, which assures permanent andunrestricted availability of the progeny of the culture of the depositto the public upon issuance of the pertinent U.S. patent or upon layingopen to the public of any U.S. or foreign patent application, whichevercomes first, and assures availability of the progeny to one determinedby the Commissioner for Patents to be entitled thereto according to 35U.S.C. § 122 and the Commissioner's rules pursuant thereto (including 37C.F.R. § 1.14 with particular reference to 866 OG 638).

The assignee of the present application has agreed that if a culture ofthe materials on deposit should die or be lost or destroyed whencultivated under suitable conditions, the materials will be promptlyreplaced on notification with another of the same. Availability of thedeposited material is not to be construed as a licensee to practice theinvention in contravention of the rights granted under the authority ofany government in accordance with the patent laws.

It will be understood that particular embodiments described herein areshown by way of illustration and not as limitations of the invention.The principal features of this invention can be employed in variousembodiments without departing from the scope of the invention. Thoseskilled in the art will recognize, or be able to ascertain using no morethan routine experimentation, numerous equivalents to the specificprocedures described herein. Such equivalents are considered to bewithin the scope of this invention and are covered by the claims.

All publications and patent applications mentioned in the specificationare indicative of the level of skill of those skilled in the art towhich this invention pertains. All publications and patent applications,and in particular relevant portions thereof, are herein incorporated byreference to the same extent as if each individual publication or patentapplication was specifically and individually indicated to beincorporated by reference.

In the claims, all transitional phrases such as “comprising,”“including,” “carrying,” “having,” “containing,” “involving,” and thelike are to be understood to be open-ended, i.e., to mean including butnot limited to. Only the transitional phrases “consisting of” and“consisting essentially of,” respectively, shall be closed orsemi-closed transitional phrases.

All of the compositions and/or methods disclosed and claimed herein canbe made and executed without undue experimentation in light of thepresent disclosure. While the compositions and methods of this inventionhave been described in terms of preferred embodiments, it will beapparent to those of skill in the art that variations may be applied tothe compositions and/or methods and in the steps or in the sequence ofsteps of the method described herein without departing from the concept,spirit and scope of the invention. More specifically, it will beapparent that certain agents which are both chemically andphysiologically related may be substituted for the agents describedherein while the same or similar results would be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

1. An antibody that binds essentially the same IFNα epitope as theanti-IFNα antibody produced by the hybridoma having ATCC Accession No.PTA-7778.
 2. The antibody of claim 1, wherein the antibody binds thesame epitope as the anti-IFNα antibody produced by the hybridoma havingATCC Accession No. PTA-7778.
 3. The antibody of claim 1, wherein theantibody selectively neutralizes a bioactivity of at least twointerferon alpha (“IFNα”) protein subtypes selected from the groupconsisting of protein subtypes A, 2, B2, C, F, G, H2, I, J1, K, 4a, 4band WA, but does not significantly neutralize at least one bioactivityof IFNα protein subtype D, wherein the bioactivity is activation of theMxA promoter or antiviral activity.
 4. The antibody of claim 1 whereinthe antibody selectively neutralizes at least eight of the IFNα proteinsubtypes.
 5. The antibody of claim 1, wherein the antibody selectivelyneutralizes a bioactivity of the IFNα protein subtypes A, 2, B2, C, F,G, H2, I, J1, K, 4a, 4b and WA, but does not significantly neutralizethe bioactivity of IFNα protein subtypes D and
 1. 6. The antibody ofclaim 1, wherein the antibody selectively neutralizes a bioactivity ofthe IFNα protein subtypes with a half maximal effective concentration(EC₅₀) selected from the group consisting of: a) less than about 300ng/ml of IFNα protein subtypes A, 2, B2, C, F, G, H2, I, J1, 4a, 4b andWA; and b) less than about 375 ng/ml of IFNα protein subtype K.
 7. Theantibody of claim 1, wherein the antibody neutralizes at least 50% ofthe bioactivity of said IFNα subtypes, wherein the bioactivity isactivation of the MxA promoter.
 8. The antibody of claim 5, wherein theantibody neutralizes at least 60% of said bioactivity.
 9. The antibodyof claim 5, wherein the antibody neutralizes at least 70% of saidbioactivity.
 10. The antibody of claim 5, wherein the antibodyneutralizes at least 80% of said bioactivity.
 11. The antibody of claim5, wherein the antibody neutralizes at least 90% of said bioactivity.12. The antibody of claim 1, wherein the antibody has an affinity forIFNα-A of at least about 1.5×10⁻⁹M.
 13. The antibody of claim 1, whereinthe antibody is a mouse antibody, a human antibody, a humanizedantibody, a chimeric antibody or an antibody fragment.
 14. The antibodyof claim 1, wherein the antibody is a monoclonal antibody.
 15. Theantibody of claim 1, wherein the antibody is a human antibody.
 16. Theantibody of claim 1, wherein the antibody is a humanized antibody. 17.The antibody of claim 16, wherein the antibody comprises a humanized orchimeric form of the monoclonal antibody ACO-2.
 18. The antibody ofclaim 1, wherein the antibody is the monoclonal antibody ACO-2.
 19. Theantibody of claim 18, wherein the antibody is a humanized form of themonoclonal antibody ACO-2.
 20. The antibody of claim 1, wherein theantibody is an antibody fragment.
 21. The antibody of claim 20, whereinthe antibody fragment comprises an Fab, Fab′, F(ab′)₂, Fv or scFvfragment.
 22. A monoclonal anti-interferon alpha (“IFNα”) antibodyproduced by the hybridoma having ATCC Accession No. PTA-7778.
 23. Ahumanized form of the monoclonal antibody of claim
 22. 24. An antibodyfragment of the monoclonal antibody of claim
 22. 25. The antibodyfragment of claim 24, wherein the antibody fragment comprises an Fab,Fab′, F(ab′)₂, Fv or scFv fragment.
 26. A host cell comprising a nucleicacid encoding the antibody of claim
 1. 27. The host cell of claim 26,wherein the host cell is a hybridoma.
 28. A hybridoma that produces anantibody of claim
 1. 29. The hybridoma of claim 28, wherein themonoclonal antibody is ACO-2
 30. The hybridoma of claim 29, wherein thehybridoma is the hybridoma having ATCC accession number PTA-7778.
 31. Acomposition comprising an antibody of any of claim
 1. 32. Apharmaceutical composition comprising the monoclonal antibody of claim 1and a pharmaceutically acceptable carrier.
 33. The pharmaceuticalcomposition of claim 32, wherein the monoclonal antibody comprises afragment of the monoclonal antibody that binds essentially the same IFNαepitope as the anti-IFNα antibody produced by the hybridoma having ATCCAccession No. PTA-7778.
 34. A pharmaceutical composition comprising afragment of the monoclonal antibody of claim 33 and a pharmaceuticallyacceptable carrier.
 35. A method for treating a disease or conditionassociated with abnormal expression of at least one IFNα protein subtypeselected from the group consisting of protein subtype A, 2, B2, C, F, G,H2, I, J1, K, 4a, 4b and WA without neutralizing IFNα protein subtypes Dbioactivity, in a subject, comprising administering to a subject havingsaid disease or condition, an effective amount of an antibody of claim1; wherein said bioactivity is activation of the MxA promoter orantiviral activity.
 36. The method of claim 35, wherein said bioactivityis MxA promoter activity.
 37. The method of claim 35, wherein thedisease or condition is selected from the group consisting of SystemicLupus Erythematosus (SLE), Graft versus Host Disease (GVHD), type 1diabetes, AIDS, lupus, psoriasis and autoimmune thyroiditis.
 38. Themethod of claim 37, wherein the disease is SLE.
 39. The method of claim37, wherein the disease is psoriasis.